Process for Synthesis of (3R,3&#39;R,6&#39;R)-Lutein and its Stereoisomers

ABSTRACT

(3R,3′R,6′R)-Lutein and (3R,3′R)-zeaxanthin are two dietary carotenoids that are present in most fruits and vegetables commonly consumed in the US. These carotenoids accumulate in the human plasma, major organs, and ocular tissues. In the past decade, numerous epidemiological and experimental studies have shown that lutein and zeaxanthin play an important role in the prevention of age-related macular degeneration (AMD) that is the leading cause of blindness in the U.S. and Western World. The invention provides a process for the synthesis of (3R,3′R,6′R)-lutein and its stereoisomers from commercially available (rac)-α-ionone by a C 15 +C 10 +C 15  coupling strategy. In addition, the present invention also provides access to the precursors of optically active carotenoids with 3-hydroxy-ε-end group that are otherwise difficult to synthesize. The process developed for the synthesis of lutein and its stereoisomers is straightforward and has potential for commercialization.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is in the field of organic chemistry. The inventionrelates to a process for the synthesis of (3R,3′R,6′R)-lutein and itsstereoisomers from commercially available (rac)-α-ionone by aC₁₅+C₁₀+C₁₅ coupling strategy. Employing this methodology,(3R,3′R,6′R)-lutein (dietary), (3R,3′S,6′S)-lutein, (3R,3′S,6′R)-lutein(3′-epilutein), and (3R,3′R,6′S)-lutein have been prepared. Based onthis strategy, the other 4 stereoisomers of lutein that are enantiomericto the above lutein isomers can also be prepared. These are:(3S,3′S,6′S)-lutein, (3S,3′R,6′R)-lutein, (3S,3′R,6′S)-lutein, and(3S,3′S,6′R)-lutein.

2. Background Art

(3R,3′R,6′R)-Lutein and (3R,3′R)-zeaxanthin are two dietary carotenoidsthat are present in most fruits and vegetables commonly consumed in theUS. These carotenoids accumulate in the human plasma, major organs, andocular tissues (macula, retinal pigment epithelium (RPE), ciliary body,iris, lens). In the past decade, numerous epidemiological andexperimental studies have shown that lutein and zeaxanthin play animportant role in the prevention of age-related macular degeneration(AMD) that is the leading cause of blindness in the U.S. and WesternWorld. While (3R,3′R)-zeaxanthin has been commercially available bytotal synthesis for more than two decades, the industrial production of(3R,3′R,6′R)-lutein by chemical synthesis has not yet materialized.Consequently, this carotenoid is commercially produced from saponifiedextracts of marigold flowers. The major difficulty with the totalsynthesis of (3R,3′R,6′R)-lutein is due to the presence of 3 stereogeniccenters at C3, C3′, and C6′ positions in this carotenoid that can resultin 8 possible stereoisomers. The chemical structures of 4 of thesestereoisomers are shown in Scheme 1. Among these, dietary(3R,3′R,6′R)-lutein (1) and one of its metabolites, (3R,3′S,6′R)-lutein(3′-epilutein) (3), have been detected in human plasma and tissues. Theother 4 stereoisomers of lutein (structures not shown), are those inwhich the configuration at C3 position is S while the stereochemistry atC3′ and C6′ remains the same as those lutein isomers shown in Scheme

To date, the only total synthesis of dietary (3R,3′R,6′R)-lutein (1) hasbeen reported by Mayer and Rüttimann (Helv. Chim. Acta, 1980,63:1451-55) and is based on the C₁₅+C₁₀+C₁₅ strategy as shown in Scheme2. According to this methodology, the C₁₅-Wittig salt,(3R)-3-hydroxy-(β-ionylideneethyl)triphenylphosphonium chloride (5), isreacted with one equiv. of 2,7-dimthylocta-2,4,6-triene-1,8-dial(C₁₀-dialdehyde) to give a C₂₅-aldehyde,(3R)-3-hydroxy-12′-apo-β-caroten-12′-al. Both starting materials forthis reaction are commercially available and have been employed in thetotal synthesis of (3R,3′R)-zeaxanthin by the same group. To completethe synthesis of (3R,3′R,6′R)-lutein, Mayer and Rüttimann prepared(3R,6R)-3-acetoxy-α-ionylideneethyl)triphenylphosphonium chloride in 8steps from (S)-4-hydroxy-2,6,6-trimethyl-2-cyclohexene-1-one in anoverall yield of 6.3%. In the final step of this synthesis, theseinvestigators reacted the C₂₅-aldehyde with(3R,6R)-3-acetoxy-α-ionylideneethyl)triphenylphosphonium chloride orbromide to obtain (3R,3′R,6′R)-lutein in 25% yield. Therefore theoverall yield for the reported total synthesis of lutein according tothis methodology was about 1.6%.

The total synthesis of lutein described in Scheme 2, involves numeroussteps and results in a poor overall yield. Consequently, this syntheticapproach does not provide an efficient and economically viable route forindustrial production of (3R,3′R,6′R)-lutein (1). Therefore, the presentinvention was developed to provide a more practical route to 1 byemploying a divergent synthetic strategy that could be simultaneouslyapplied to the synthesis of other stereoisomers of this carotenoid suchas (3R,3′S,6′S)-lutein (2), (3R,3′S,6′R)-lutein (3), and(3R,3′R,6′S)-lutein (4). In addition, this synthetic strategy alsoprovides access to the precursors of optically active carotenoids with3-hydroxy-ε-end group that are otherwise difficult to prepare.

SUMMARY OF THE INVENTION

Despite the difficulties encountered with the synthesis of(3R,3′R,6′R)-lutein, the C₁₅+C₁₀+C₁₅ building block strategy for thesynthesis of carotenoids is, in most cases, the method of choice. Thisis because the formation of the double bonds at 11 and 11′ positionsyields predominantly the all-E (trans)-isomer (Soukup, M; Spurr, P;Widmer E. In: Carotenoids, Volume 2: Synthesis, Britton, G;Liaaen-Jensen, S; Pfander, H. Eds.; Birkhä user: Basel, 1995, pp 7-14).Therefore, this strategy has also been employed in the presentinvention. However, because of the poor overall yield in the reportedsynthetic strategy by Mayer and Rüttimann, we employed entirelydifferent C₁₅- and C₁₀-building blocks. This was because(3R,6R)-3-acetoxy-α-ionylideneethyl)triphenylphosphonium halide that wasused in the final step of the reported synthesis of lutein appeared tobe difficult to synthesize due to the presence of an acid-sensitiveallylic hydroxyl group in the precursor to this Wittig salt (Scheme 2).In addition, the olefination of (3R)-3-hydroxy-12′-apo-β-caroten-12′-al(C₂₅-aldehyde) with this Wittig salt according to Mayer and Rüttimannonly gave 25% yield of lutein.

The retrosynthetic pathways employed in the present invention is shownin Scheme 3. In contrast to the reported synthesis of lutein, the finalstep of our synthesis involved the elongation of the optically pureC₂₅-hydroxyaldehydes 6-9 with the Wittig salt 5 that could be readilyprepared according to the known processes (Widmer et al., Helv. Chim.Acta, 1990, 73: 861-867; Soukup et al., Helv. Chim. Acta, 1990, 73:868-873). We rationalized that the optically pure C₂₅-hydroxyaldehydes6-9 could be prepared from deprotection of their correspondingdimethylacetals 10-13 under mild acidic conditions without epimerizationof their allylic hydroxyl groups at C3. These acetals could in turn beprepared from the reaction of protected Wittig salt 14 with theoptically pure C₁₅-hydroxyaldehydes 15-18 with the requiredstereochemistry at C3 and C6. The protected Wittig salt 14 was readilyaccessible according to published methods (Bernhard et al., Helv. Chim.Acta, 1980, 63:1473-1490; Haugen, Acta Chimica Scand. 1994, 48:657-664). The application of this Wittig salt in the synthesis ofunsymmetrical carotenoids with sensitive end-groups has been welldocumented in the literature (Bernhard et al., Helv. Chim. Acta, 1980,63:1473-1490; Haag and Eugster, Helv. Chim. Acta, 1985, 68:1897-1906;Yamano et al. Heterocycles, 2000, 52: 141-146). However, this buildingblock has not been employed in the synthesis of lutein or itsprecursors. The C₁₅-hydroxynitriles 19-22 as a racemic mixture or withthe appropriate stereochemistry at C3 and C6 could serve as theprecursors to C₁₅-hydroxyaldehydes 15-18.(7E,9E)-3-Keto-α-ionylideneacetonitrile (23a) and its (7E,9Z)-isomer(23b), prepared from nitriles 24a and 24b, could be transformed intoC₁₅-hydroxynitriles 19-22. However the (7E,9E)-isomer (23a) would bepreferable since this would avoid the difficulties associated with theseparation of optically active E/Z-isomers throughout our entiresynthetic strategy.

The commercially available and inexpensive (rac)-α-ionone was selectedas the starting material for the synthesis of nitriles 24a/24b that havebeen previously synthesized according to known methods. However, we hadto develop a methodology that could provide 24a as a single isomer andtransform this nitrile into 23a without stereisomerization. Otherchallenges with our synthetic approach involved separation ofC₁₅-hydroxyaldehydes 15-18 and their precursors in high optical purityand maintaining their integrity throughout the total synthesis ofluteins 1-4. It should be noted that all of the precursors to luteins1-4 that are shown in our retrosynthetic pathways in Scheme 3, arereported here for the first time and have not been synthesizedpreviously. This is with the exception of nitriles 24a/24b andketonitriles 23a/23b that have been prepared as a mixture of E/Z isomersby entirely different processes than those developed in the presentinvention.

One of the key starting materials in the retrosynthetic pathways shownin Scheme 3 is (rac)-3-keto-α-ionylideneacetonitrile which had to bepreferentially synthesized as the (7E,9E)-isomer (23a) at the expense ofits (7E,9Z)-isomer (23b). This is because when (rac)-ketonitrile 23a isreduced in the following step, a new stereogenic center at C3 isgenerated that results in the formation of four stereoisomers, namely,(rac)-hydroxynitriles 19-22. Consequently, the reduction of a mixture ofketonitriles 23a and 23b, could afford as many as 8 stereoisomerichydroxynitriles which would be difficult to separate in high opticallypurity. Therefore, the initial goal of this invention was to explore thepossible routes by which (rac)-α-ionone could be transformed intoketonitrile 23a. Three synthetic routes were employed for transformationof (rac)-α-ionone to ketonitrile 23a that served as a precursor toC₁₅-hydroxynitriles 19-22 (Scheme 4). According to the first route,Horner-Wadsworth-Emmons (HWE) reaction of (rac)-α-ionone with diethylcyanomethylphosphonate or diisopropyl cyanomethylphosphonate gave(rac)-α-ionylideneacetonitriles 24a (75%) and 24b (25%) as a mixture ofisomers that were converted to a mixture of 23a (75%) and 23b (25%) byallylic oxidation.

However, a more effective strategy (Route 2, Scheme 4) was developedthat involved Knoevenagel condensation of (rac)-α-ionone withcyanoacetic acid to afford 24a (92%) as the major isomer and 24b (8%) asthe minor isomer. When a mixture of 24a (92%) and 24b (8%) was subjectedto allylic oxidation, 23a (92%) and 23b (8%) were obtained withoutE/Z-isomerization and the (7E,9E)-isomer (23a) could be crystallizedfrom the mixture.

In an alternative approach (Route 3, Scheme 4), (rac)-α-ionone was firstconverted to (rac)-3-keto-α-ionone by allylic oxidation followed by HWEolefination with diethyl cyanomethylphosphonate to yield a mixture of23a (75%) and 23b (25%). Consequently, among these three strategies,Route 2 that involved Knoevenagel reaction of (rac)-α-ionone withcyanoacetic acid and provided 23a in high stereoselectivity was thepreferred route. Reduction of the ketonitrile 23a with a number ofreducing agents provided a mixture of four stereoisomericC₁₅-hydroxynitriles 19-22. Among the reducing agents employed, potassiumtri-sec-butylborohydride (K-SELECTRIDE™) at −30° C. in TBME or THFproduced the greatest amount of the hydroxynitriles 19 and 20 (86%)relative to the hydroxynitriles 21 and 22 (14%). However, whenBH₃/(R)-2-methyl-CBS-oxazaborolidine was used as the reducing agent,this stereoselectivity was reversed and hydroxynitriles 21 and 22 (86%)were the major products and hydroxynitriles 19 and 20 (14%) were theminor products. The separation of hydroxynitriles 19 and 20 fromhydroxynitriles 21 and 22 by column chromatography proved to bechallenging. However, this was accomplished by subjecting these nitrilesto two successive column chromatography separations. In the next step,enzyme-mediated acylation with lipase AK (Pseudomonas fluorescens) orlipase PS (Pseudomonas cepacia) was employed to separate theenantiomeric hydroxyaldehydes 21 from 22. However, these enzymaticseparations resulted in poor enantiomeric excess (ee) and the partiallyresolved enantiomers had to be subjected to a second enzymatic acylationto provide the optically pure hydroxynitriles 19-22. Consequently, thisapproach was not appealing due to the need for repeated columnchromatography and enzyme-mediated acylation of racemic nitriles.Therefore, a more robust strategy was developed that eliminated thesedifficulties and afforded the hydroxyaldehydes 15-18 in excellentoptical purities (Scheme 5). As shown in Scheme 5, the hydroxynitriles19-22 were first transformed into a racemic mixture of hydroxyaldehydes15-18 by DIBAL-H and the mixture was then subjected to columnchromatography. Unlike hydroxynitriles, hydroxyaldehydes 15+16 werereadily separated from hydroxyaldehydes 17+18 by column chromatography.In an alternative one-pot reaction, ketonitrile 23a was reduced tohydroxynitriles 19-22 with K-SELECTRIDE™followed by the reduction withDIBAL-H to afford hydroxyaldehydes 15-18 in one convenient step.

In the following step, enzyme-mediated acylation was employed to resolvethe racemic mixture of hydroxyaldehydes 15 and 16. Therefore, when amixture of hydroxyaldehydes 15 and 16 was subjected to enzymaticacylation with lipase AK (Pseudomonas fluorescens) in the presence ofvinyl acetate in refluxing pentane (35-36° C.), hydroxyaldehyde 16 wasacylated to acetoxyaldehyde 25 within 48 h, while hydroxyaldehyde 15remained unchanged (Scheme 5). Acetoxyaldehydes 25 was then readilyseparated from hydroxyaldehyde 15 by column chromatography.Acetoxyaldehyde 25 was saponified with KOH/MeOH at 0° C. to yieldhydroxyaldehyde 16. According to this strategy, hydroxyaldehydes 15 and16 were obtained in enantiomeric excess (ee) of 94% and 93%,respectively.

Similarly, the resolution of a racemic mixture of hydroxyaldehydes 17and 18 was accomplished with enzyme-mediated acylation with lipase AK(Pseudomonas fluorescens) in the presence of vinyl acetate in refluxingpentane. While hydroxyaldehyde 17 underwent acylation to acetoxyaldehyde26, hydroxyaldehyde 18 remained unchanged; these were then separated bycolumn chromatography. Saponification of acetoxyaldehyde 26 withKOH/MeOH at 0° C. afforded hydroxyaldehyde 17. Employing thismethodology, hydroxyaldehydes 17 and 18 were obtained in enantiomericexcess (ee) of 91% and 92%, respectively. Therefore, all fourC₁₅-hydroxyaldehydes 15-18 became accessible in excellent optical purityand were utilized in the synthesis of luteins 1-4 according to thesynthetic pathways shown in Scheme 6.

The Wittig reaction of optically pure C₁₅-hydroxyaldehydes 15-18 withthe required stereochemistry at C3 and C6 with the protected Wittig salt14 afforded 3-hydroxy-12′-apo-ε-caroten-12′-al dimethylacetals 10-13(protected C₂₅-aldehydes) in yields ranging from 75-85%. As part of thework-up of the same reaction, the protecting group in acetals 10-13 wasremoved under mild acidic conditions without epimerization at C3 toafford C₂₅-hydroxyaldehydes 6-9, respectively. In the final step of thesynthesis of luteins, aldehydes 6-9 were allowed to react with theWittig salt 5 to yield luteins 1-4 in yields ranging from 65-74%.Therefore, according to the present invention, luteins 1 and 2 were eachprepared in an overall yield of 21% based on the optically activeC₁₅-hydroxyaldehydes 15+16. Similarly, luteins 3 and 4 were prepared inoverall yields of 16% and 18%, respectively. These C₁₅-hydroxyaldehydesserved as the key starting material in our synthetic strategy.

In one embodiment of the present invention, a compound having theFormula (I):

is synthesized by reacting a compound having the Formula (II):

with a compound having the Formula (III):

via Wittig coupling, wherein A⊖ is an anionic counterion such as Cl⁻,Br⁻or I⁻. In some embodiments, the compound of Formula (I) is(3R,3′R,6′R)-lutein, (3R,3′S,6′S)-lutein, (3R,3′S,6′R)-lutein,(3R,3′R,6′S)-lutein, (3S,3′S,6′S)-lutein, (3S,3′R,6′R)-lutein,(3S,3′R,6′S)-lutein or (3S,3′S,6′R)-lutein, or a combination thereof. Insome embodiments, the compound of Formula (III) is(3R)-3-hydroxy-(β-ionylideneethyl)triphenylphosphonium salt or(3S)-3-hydroxy-(β-ionylideneethyl)triphenylphosphonium salt. In someembodiments, the triphenylphosphonium salt is a fluoride, chloride,bromide or iodide salt.

In one embodiment, the compound having the Formula II is prepared bydeprotecting a compound having the Formula (IV):

to obtain the compound having the Formula II, wherein R¹ and R² areindependently a branched C₁-C₇ alkyl, a straight chain C₁-C₇ alkyl, ortaken together form a 5-7 membered ring. In some embodiments, R¹ and R²are independently C₁-C₇ alkyl. In some embodiments, R¹ and R² aremethyl. In some embodiments, the compound having the Formula (IV) isdeprotected under mild acidic conditions without loss of optical purity.In some embodiments, the compound having the Formula (II) is(3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (6),(3S,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (7),(3S,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (8) or(3R,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (9), or a combinationthereof. In some embodiments:

-   -   (i) (3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al        dimethylacetal (10) is deprotected to form        (3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (6),    -   (ii) (3S,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al        dimethylacetal (11) is deprotected to form        3S,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (7);    -   (iii) (3S,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al dimethyl        acetal (12) is deprotected to form        (3S,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (8) or    -   (iv) (3R,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al        dimethylacetal (13) is deprotected to form        (3R,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (9).

In one embodiment, a compound having the Formula (IV) is prepared byelongating a compound having the Formula V:

with a compound having the Formula VI:

via Wittig coupling to obtain the compound having the Formula IV, whereX⊖ is an anionic counterion such as Cl⁻, Br⁻ or I⁻, wherein R³ and R⁴are independently a branched C₁-C₇ alkyl, a straight chain C₁-C₇ alkyl,or taken together form a 5-7 membered ring. In some embodiments, R¹ andR² are independently C₁-C₇ alkyl. In some embodiments, R¹ and R² aremethyl. In some embodiments, the compound having the Formula (IV) isprotected C₂₅-hydroxyaldehyde 10, 11, 12, or 13, or a combinationthereof. In some embodiments, the compound having the Formula (VI) is(all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium salt.In some embodiments, the triphenylphosphonium salt is a chloride,bromide or iodide salt. In another embodiment:

-   -   (i)        (all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium        salt is reacted with        (7E,9E,3R,6R)-3-hydroxy-α-ionylideneacetaldehyde (15) to obtain        protected C₂₅-hydroxyaldehyde 10;    -   (ii)        (all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium        salt is reacted with        (7E,9E,3S,6S)-3-hydroxy-α-ionylideneacetaldehyde (16) to obtain        protected C₂₅-hydroxyaldehyde 11;    -   (iii)        (all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium        salt is reacted with        (7E,9E,3S,6R)-3-hydroxy-α-ionylideneacetaldehyde (17) to obtain        protected C₂₅-hydroxyaldehyde 12; or    -   (iv)        (all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium        salt is reacted with        (7E,9E,3R,6S)-3-hydroxy-α-ionylideneacetaldehyde (18) to obtain        protected C₂₅-hydroxyaldehyde 13.

In one embodiment, the compound having the Formula V:

is prepared by reacting the cyano group of a compound having the FormulaVII:

with a reducing agent to obtain the compound having the Formula V. Insome embodiments, the compound having the Formula (V) isC₁₅-hydroxyaldehyde 15, 16, 17 and 18, or a combination thereof. In someembodiments, the compound having the Formula (VII) is(3R,6R)-3-hydroxy-α-ionylideneacetonitrile (19),(3S,6S)-3-hydroxy-α-ionylideneacetonitrile (20),(3S,6R)-3-hydroxy-α-ionylideneacetonitrile (21) or(3R,6S)-3-hydroxy-α-ionylideneacetonitrile (22), or a combinationthereof.

In one embodiment, a mixture of C₁₅-hydroxynitriles(3R,6R)-3-hydroxy-α-ionylideneacetonitrile (19),(3S,6S)-3-hydroxy-α-ionylideneacetonitrile (20),(3S,6R)-3-hydroxy-α-ionylideneacetonitrile (21) and(3R,6S)-3-hydroxy-α-ionylideneacetonitrile (22) is reduced withdiisobutylaluminum hydride (DIBAL-H), to obtain a mixture ofC₁₅-hydroxyaldehydes 15, 16, 17 and 18.

In some embodiments, a mixture of C₁₅-hydroxyaldehydes 15, 16, 17 and 18is separated by using a combination of column chromatography andenzyme-mediated acylation. In some embodiments, a mixture ofC₁₅-hydroxyaldehydes 15 and 16 is separated from the mixture ofC₁₅-hydroxyaldehyde 15, 16, 17 and 18 by column chromatography using acombination of a hydrocarbon solvent selected from the group consistingof pentane, hexane, heptane and cyclohexane, and ethyl acetate oracetone, to obtain a mixture of C₁₅-hydroxyaldehydes 15 and 16. In someembodiments, the column chromatography is carried out on n-silica.

In some embodiments, the mixture of C₁₅-hydroxyaldehydes 15 and 16 isacylated with lipase AK (Pseudomonas fluorescens) or lipase PS(Pseudomonas cepacia) in the presence of an acyl donor such as vinylacetate, wherein C₁₅-hydroxyaldehyde 16 is converted to(3S,6S)-3-acetoxy-α-ionylideneacetaldehyde (25) whileC₁₅-hydroxyaldehyde 15 remains unesterified. In some embodiments,C₁₅-acetoxyaldehyde 25 is saponified with alcoholic potassium hydroxide(KOH) or sodium hydroxide (NaOH) to obtain C₁₅-hydroxyaldehyde 16.

In some embodiments, a mixture of C₁₅-hydroxyaldehydes 17 and 18 isseparated from the mixture of C₁₅-hydroxyaldehyde 15, 16, 17 and 18 bycolumn chromatography using a combination of a hydrocarbon solventselected from the group consisting of pentane, hexane, heptane andcyclohexane, and ethyl acetate or acetone, to obtain a mixture ofC₁₅-hydroxyaldehydes 17 and 18. In some embodiments, the columnchromatography is carried out on n-silica. In some embodiments, themixture of C₁₅-hydroxyaldehydes 17 and 18 is acylated with lipase AK(Pseudomonas fluorescens) or lipase PS (Pseudomonas cepacia) in thepresence of an acyl donor such as vinyl acetate, whereinC₁₅-hydroxyaldehyde 17 is converted to(3S,6R)-3-acetoxy-α-ionylideneacetaldehyde (26) whileC₁₅-hydroxyaldehyde 18 remains unesterified. In some embodiments,C₁₅-acetoxyaldehyde 26 is saponified with alcoholic potassium hydroxide(KOH) or sodium hydroxide (NaOH) to obtain C₁₅-hydroxyaldehyde 17.

In one embodiment, the ketone group of a compound having the Formula(VIII):

-   -   is reacted with a reducing agent, to obtain the compound having        the Formula (VII):

In some embodiments, the reducing agent is stereoselective. In someembodiments, a compound having the Formula (VIII) is(7E,9E)-3-keto-α-ionylideneacetonitrile (23a) or(7E,9Z)-3-keto-α-ionylideneacetonitrile (23b), or a combination thereof.In some embodiments, the compound having the Formula (VII) is(3R,6R)-3-hydroxy-α-ionylideneacetonitrile (19),(3S,6S)-3-hydroxy-α-ionylideneacetonitrile (20),(3S,6R)-3-hydroxy-α-ionylideneacetonitrile (21) or(3R,6S)-3-hydroxy-α-ionylideneacetonitrile (22), or a combinationthereof.

In some embodiments, (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) isstereoselectively reduced with a reducing agent to obtain(3,6-trans)-C₁₅-hydroxynitriles 19+20 and (3,6-cis)-C₁₅-hydroxynitriles21+22 in a ratio ranging from 6:1 to 1:6. In some embodiments, thereducing agent is NaBH₄, NaBH₄/dl-tartaric acid, NaBH₄/d-tartaric acid,NaBH₄/l-tartaric acid, NaBH₄/dibenzoyl-d-tartaric acid,NaAlH₂(OCH₂CH₂OMe)₂ (RED-AL™), LiB[CHMeCH₂CH₃]₃H (L-SELECTRIDE™),NaB[CHMeCH₂CH₃]₃H(N-SELECTRIDE™), KB[CHMeCH₂CH₃]₃H (K-SELECTRIDE™),KB[CHMeCHMe₂]₃H (KS-SELECTRIDE™), BH₃/(R)-2-methyl-CBS-oxazaborolidine,or BH₃/(S)-2-methyl-CBS-oxazaborolidine.

In some embodiments, ketonitrile 23a is selectively reduced withKB[CHMeCH₂CH₃]₃H (K-SELECTRIDE™) to obtain(3,6-trans)-C₁₅-hydroxynitriles 19+20 as the major products and(3,6-cis)-C₁₅-hydroxynitriles 21+22 as the minor products.

In some embodiments, ketonitrile 23a is selectively reduced withBH₃/(R)-2-methyl-CBS-oxazaborolidine to obtain(3,6-cis)-C₁₅-hydroxynitriles 21+22 as the major products and(3,6-trans)-C₁₅-hydroxynitriles 19+20 as the minor products. In someembodiments, ketonitrile 23a is reduced to obtain a mixture ofC₁₅-hydroxyaldehydes 15, 16, 17 and 18 by (i) reducing(7E,9E)-3-keto-α-ionylideneacetonitrile (23a) with a metal hydridereagent to form a mixture of C₁₅-hydroxynitriles 19, 20, 21 and 22 and(ii) reducing the mixture of C₁₅-hydroxynitriles 19, 20, 21 and 22 withDIBAL-H to obtain a mixture of C₁₅-hydroxyaldehydes 15, 16, 17 and 18 ina one-pot reaction. In some embodiments, the metal hydride reagent isNaAlH₂(OCH₂CH₂OMe)₂ (RED-AL™), LiB[CHMeCH₂CH₃]₃H (L-SELECTRIDE™),NaB[CHMeCH₂CH₃]₃H (N-SELECTRIDE™), KB[CHMeCH₂CH₃]₃H (K-SELECTRIDE™) orKB[CHMeCHMe₂]₃H (KS-SELECTRIDE™).

In one embodiment, a compound having the Formula (VIII):

is prepared by reacting a compound having the Formula (IX):

with an oxidizing agent, to obtain the compound having the Formula(VIII) via allylic oxidation. In some embodiments, the compound havingthe Formula (IX) is (7E,9E)-α-ionylideneacetonitrile (24a) or(7E,9Z)-α-ionylideneacetonitrile (24b), or a mixture thereof. In someembodiments, the compound having the Formula (VIII) is(7E,9E)-3-keto-α-ionylideneacetonitrile (23a) or(7E,9Z)-3-keto-α-ionylideneacetonitrile (23b), or a combination thereof.

In some embodiments, a mixture of (7E,9E)-α-ionylideneacetonitrile (24a)and (7E,9Z)-α-ionylideneacetonitrile (24b) in an isomeric ratio rangingfrom 3:1 to 12:1 is reacted with an oxidizing reagent, to obtain amixture of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) and(7E,9z)-3-keto-α-ionylideneacetonitrile (23b) in an isomeric ratioranging from 3:1 to 12:1.

In some embodiments, a mixture of(7E,9E)-3-keto-α-ionylideneacetonitrile (23a) and(7E,9Z)-3-keto-α-ionylideneacetonitrile (23b) is purified by separatinga mixture of ketonitrile 23a and ketonitrile 23b via crystallizationwith an alcohol such as ethanol, at a temperature ranging from −15 to 0°C.

In some embodiments, the compound having the Formula (IX) is a mixtureof (7E,9E)-α-ionylideneacetonitrile (24a) and(7E,9Z)-α-ionylideneacetonitrile (24b) in an isomeric ratio ranging from3:1 to 12:1 is oxidized with a combination of tert-BuOOH (TBHP) andbleach (5.25% NaOCl), at a temperature ranging from −5 to 0° C., in asolvent selected from the group consisting of acetonitrile (CH₃CN),methylene chloride (CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran(THF), tert-butyl methyl ether (TBME), dimethylformamide (DMF),dimethylsulfoxide (DMSO), ethylene glycol, a straight chain C₁-C₅alcohol and a branched C₁-C₅ alcohol, to obtain the compound having theFormula (VIII) as a mixture of (7E,9E)-3-keto-α-ionylideneacetonitrile(23a) and (7E,9Z)-3-keto-α-ionylideneacetonitrile (23b).

In some embodiments, the compound having the Formula (IX) is a mixtureof (7E,9E)-α-ionylideneacetonitrile (24a) and(7E,9Z)-α-ionylideneacetonitrile (24b) in an isomeric ratio ranging from3:1 to 12:1 and is oxidized with a combination of tert-BuOOH (TBHP) andPd/C at a temperature ranging from 0° C. to room temperature (R.T.), ina solvent selected from the group consisting of acetonitrile (CH₃CN),methylene chloride (CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran(THF), tert-butyl methyl ether (TBME), dimethylformamide (DMF),dimethylsulfoxide (DMSO), ethylene glycol, a straight chain C₁-C₅alcohol and a branched C₁-C₅ alcohol, to obtain the compound having theFormula (VIII) as a mixture of (7E,9E)-3-keto-α-ionylideneacetonitrile(23a) and (7E,9Z)-3-keto-α-ionylideneacetonitrile (23b).

In some embodiments, a compound having the Formula (IX):

by condensing a compound having the Formula (X):

with cyanoacetic acid to obtain the compound having the Formula (IX). Insome embodiments, the compound having the Formula (IX) is(7E,9E)-α-ionylideneacetonitrile (24a) or(7E,9Z)-α-ionylideneacetonitrile (24b), or a combination thereof. Insome embodiments, the compound having the Formula (X) is (rac)-α-ionone.

In one embodiment, (rac)-α-ionone is condensed with cyanoacetic acid inthe presence of an amine such as cyclohexylamine, at a temperatureranging from 80° C. to 100° C., to obtain(7E,9E)-α-ionylidene-acetonitrile (24a) and(7E,9Z)-α-ionylideneacetonitrile (24b) in a ratio of 12:1 or greater. Insome embodiments, the mixture of nitriles 24a and 24b in an isomericratio of 12:1 or greater is purified by vacuum distillation, wherein theisomeric ratio of 24a and 24b is unaltered.

In one embodiment, (rac)-3-keto-α-ionone is prepared by reacting(rac)-α-ionone with an oxidizing agent to obtain (rac)-3-keto-α-ionone.

In some embodiments, (rac)-α-ionone is reacted with a combination oftert-BuOOH (TBHP) and bleach, at a temperature ranging from −5 to 0° C.,in a solvent selected from the group consisting of acetonitrile (CH₃CN),methylene chloride (CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran(THF), tert-butyl methyl ether (TBME), dimethylformamide (DMF),dimethylsulfoxide (DMSO), ethylene glycol, a straight chain C₁-C₅alcohol and a branched C₁-C₅ alcohol, to obtain to(rac)-3-keto-α-ionone.

In some embodiments, (rac)-α-ionone is reacted with a combination oftert-BuOOH (TBHP) and Pd/C, at a temperature ranging from 0° C. to roomtemperature (R.T.), in a solvent selected from the group consisting ofacetonitrile (CH₃CN), methylene chloride (CH₂Cl₂), ethyl acetate,hexane, tetrahydrofuran (THF), tert-butyl methyl ether (TBME),dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethylene glycol, astraight chain C₁-C₅ alcohol and a branched C₁-C₅ alcohol, to obtain to(rac)-3-keto-α-ionone. In some embodiments, ketonitriles 23a and 23b areprepared by condensing (rac)-3-keto-α-ionone with (EtO)₂P(O)CH₂CN or(iso-PrO)₂P(O)CH₂CN in the presence of a base to obtain ketonitriles 23aand 23b.

In one embodiment, a compound of the Formula XII is prepared byoxidatively degrading a compound having the Formula XI:

with an oxidizing agent, to obtain a compound of the Formula XII:

and a compound of the Formula XIII:

In some embodiments, the compound having the Formula (XI) is(3R,3′R,6′R)-lutein diacetate, (3R,3′S,6′S)-lutein diacetate,(3R,3′S,6′R)-lutein diacetate, (3R,3′R,6′S)-lutein diacetate,(3S,3′S,6′S)-lutein diacetate, (3S,3′R,6′R)-lutein diacetate,(3S,3′R,6′S)-lutein diacetate or (3S,3′S,6′R)-lutein diacetate or acombination thereof.

In some embodiments, (3R,6R)-3-hydroxy-13-apo-ε-caroten-13-one (27) and(3R)-3-hydroxy-13-apo-β-caroten-13-one (28) are prepared by oxidativelydegrading (3R,3′R,6′R)-lutein diacetate with tert-BuOOH (TBHP) andbleach, at a temperature ranging from −5° C. to room temperature (R.T.),in a solvent selected from the group consisting of acetonitrile (CH₃CN),methylene chloride (CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran(THF), tert-butyl methyl ether (TBME), dimethylformamide (DMF),dimethylsulfoxide (DMSO), a straight chain C₁-C₅ alcohol and a branchedC₁-C₅ alcohol to obtain (3R,6R)-3-hydroxy-13-apo-ε-caroten-13-one (27)and (3R)-3-hydroxy-13-apo-β-caroten-13-one (28).

DETAILED DESCRIPTION OF THE INVENTION

All chemicals and reagents were commercially available and obtained fromAldrich Chemical Co. (St. Louis, Mo.). Lipase AK (pseudomonasfluorescens) and Lipase PS (Pseudomonas cepacia) were from Amano EnzymeUSA (Lombard, Ill.). All carotenoids and their precursors were fullycharacterized by ¹H and ¹³C-NMR, MS, and UV-Vis, and circular dichroism(CD). Combination of NMR and CD was employed to assign the relative andabsolute stereochemistry of all synthetic carotenoids and theirprecursors. The purity of all compounds was determined by HPLC on asilica-based nitrile bonded column (hexane, 75%; CH₂Cl₂ 25%; MeOH, 0.5%;0.7 mL/min) and a chiral HPLC [amylosetris-(3,5-dimethylphenylcarbamate)] column was employed to assess theoptical purity of stereoisomers. The absolute configurations ofC₁₅-hydroxynitriles 19-22 and C₁₅-hydroxyaldehydes 15-18 wereunequivocally established by comparison of their NMR and CD spectra withthose of (3R,6R)-3-hydroxy-13-apo-ε-caroten-13-one which was preparedfrom oxidative degradation of naturally occurring (3R,3′R,6′R)-lutein.

Synthesis of (7E,9E)-3-Keto-α-ionylideneacetonitrile (23a) from(rac)-α-Ionone. In one embodiment of the present invention, HWE reactionof commercially available (rac)-α-ionone with diethylcyanomethylphosphonate in dry tert-butyl methyl ether (TBME) ortetrahydrofuran (THF) using NaH or NaOMe/MeOH as base gave(7E,9E)-nitrile 24a (75%) and (7E,9Z)-nitrile 24b (25%) in 74% isolatedyield after distillation (Route 1, Scheme 4). Alternatively, diisopropylcyanomethylphosphonate could also be used with similar results. Amixture of nitriles 24a and 24b was then oxidized with tert-BuOOH (TBHP,70% in water), household bleach, and catalytic amounts of K₂CO₃ inacetonitrile at −5 to 0° C. to yield ketonitriles 23a (75%) and 23(25%). After purification by chromatography, a mixture of these nitrileswas obtained in 57% yield. This mixture was crystallized from ethanol at−15° C. to give the (7E,9E)-ketonitrile 23a as a white crystal free from23b in 37% isolated yield. While this reaction can be carried out inother solvents such as ethyl acetate, ethylene glycol, and hexane, thehighest isolated yield of 57% was obtained with acetonitrile andethanol. This water-based oxidation system, using household laundrybleach and aqueous TBHP, has been shown to convert steroidal olefins toα,β-enones by an economical and environmentally friendly methodology(Marwah, Green Chem., 2004, 6, 570-577. Ketonitriles 23a and 23b werealso prepared in 53% yield by palladium(II)-mediated oxidation ofnitriles 24a and 24b with TBHP in dichloromethane (CH₂Cl₂) at 0° C.similar to a methodology that has been employed for allylic oxidation ofolefins (Yu and Corey, Org. Lett. 2002, 4: 2727-2730). However to date,there are no literature reports on the direct oxidation of nitriles 24aand 24b to ketonitriles 23a and 23b. These oxidation reactions clearlyrevealed that conversion of a mixture of 24a/24b to 23a/23b is notaccompanied by E/Z-isomerization and the isomeric ratio of thesenitriles remains unchanged. As mentioned earlier, the reduction of amixture of ketonitriles 23a and 23b can yield a complicated mixture of(7E,9E)- and (7E,9Z)-hydroxynitriles 19-22 that would be difficult toseparate in high optical purity (Scheme 4). Therefore, an alternativeprocess was needed that could preferably provide 23a or its precursor24a as a single isomer. It has been previously shown that Knoevenagelcondensation of β-ionone with cyanoacetic acid in boiling pyridine (115°C.) in the presence of catalytic amounts of piperidinium acetate affordsβ-ionylideneacetonitrile in 75% yield, predominantly as the(7E,9E)-isomer (Andriamialisoa et al. Tetrahedron Lett., 1993, 34:8091-8092). However, in this literature report, the isomeric ratio of(7E,9E)/(7E,9Z) was not specified. When we applied the reported reactionconditions employed with β-ionone to condensation of (rac)-α-ionone withcyanoacetic acid, no reaction was observed. After examining thisreaction with a number of organic amines, we discovered thatcyclohexylamine could promote this reaction under mild conditions togive a high yield of (7E,9E)-α-ionylideneacetonitrile (24a) (Route 2,Scheme 4).

Therefore, in a preferred embodiment, Knoevenagel condensation of(rac)-α-ionone (1 eq) with cyanoacetic acid (1.3 eq) in cyclohexylamine(3 eq), also used as solvent, at 80-85° C. after 3.5 h affords 24a (92%)and 24b (8%) as a colorless oil in 75% isolated yield afterdistillation. Another reported method for the synthesis ofα-ionylideneacetonitrile and β-ionylideneacetonitrile, involvescondensation of α-ionone or β-ionone with methyl cyanoacetate in thepresence of glacial acetic acid, acetamide, and ammonium acetate toyield the corresponding methyl α-ionylidenecyanoacetate or methylβ-ionylidenecyanoacetate (Young et al. J. Am. Chem. Soc., 1944, 66:520-524). These esters were then saponified to their corresponding α- orβ-ionylidenecyanoacetic acid and subsequently decarboxylated to α- orβ-ionylideneacetonitrile. Due to the old nature of this publication andlack of sophisticated analytical methods in 1944, the ratio of(7E,9E)/(7E,9Z) isomers in these nitriles were not reported.

In the following step, the mixture of 24a:24b=92%:8% (1 eq) and K₂CO₃(0.1 eq) in acetonitrile (16 eq) was oxidized with tert-BuOOH (TBHP, 70%in water, 7 eq) and household bleach containing 5.25% NaOCl (2 eq ofNaOCl) at −5 to 0° C. under nitrogen to yield ketonitriles 23a (92%) and23b (8%). After extraction with ethyl acetate (EtOAc), the product wasthen purified by column chromatography (n-Silica) employing hexane:EtOAc(90%:10 to 70%:30%) to give a mixture of 23a (92%) and 23b (8%) in 53%yield. When this mixture was dissolved in ethanol and cooled down to−15° C., (7E,9E)-ketonitrile 23a was obtained as white crystals in 37%isolated yield and contained no measurable amounts of 23b. Therefore thepresent invention relates to two novel routes that converts(rac)-α-ionone to a single isomer of(7E,9E)-3-keto-α-ionylideneacetonitrile (23a) in crystalline form viaallylic oxidation of α-ionylideneacetonitriles 24a and 24b. Depending onthe selected route, the isomeric (7E,9E):(7E,9Z) ratio of nitriles24a:24b may vary from 75%:25% to 92%:8% (Routes 1 and 2, Scheme 4).

In an alternative embodiment (Route 3, Scheme 4), (rac)-α-ionone wasoxidized to crystalline (rac)-3-keto-α-ionone with TBHP (70% in water),bleach, and catalytic amounts of K₂CO₃ in ethyl acetate at −5 to 0° C.in 64% isolated yield. The palladium(II)-mediated oxidation of(rac)-α-ionone with TBHP in CH₂Cl₂ also afforded this ketone as a whitecrystalline solid in 53% isolated yield. There are three reportedprocedures for preparation of (rac)-3-keto-α-ionone in the literature.The first procedure employs tert-butyl chromate to oxidize(rac)-α-ionone to (rac)-3-keto-α-ionone in only 14% isolated yield(Prelog and Osgan, Helv. Chim. Acta, 1952, 35: 986-992) and the seconduses Ac₂Co.4H₂O/NH₄Br/O₂ to improve the yield to 31% (Widmer et al.,Helv. Chim. Acta 1982, 65: 944-57). More recently, another procedure forallylic oxidation of ionone-like dienes with TBHP catalyzed by CaCl₂ andMgCl₂.6H₂O at 60° C. has also been reported that can afford(rac)-3-keto-α-ionone in yields comparable to ours (Yang et al. Synlett2006, 16: 2617-2620).

The HWE reaction of (rac)-3-keto-α-ionone with diethylcyanomethylphosphonate in TBME or THF gave (rac)-ketonitrile 23a (75%)and 23b (25%) in 81% yield. After purification by flash chromatographyand crystallization from ethanol at −15° C., (7E,9E)-ketonitrile 23a wasobtained as white crystals in 40% isolated yield. This reaction has beenpreviously reported by Imai et al. to yield a mixture of 23a and 23b asan oil that was not crystallized and the isomeric ratio of theseketonitriles were not reported (Imai, Photochem. Photobiol. 1999, 70:111-115). Our methodology for the synthesis of ketonitriles 23a and 23baccording to the routes 1 and 2, as shown in Scheme 5, is novel and hasnot been reported previously. Further, the present invention, for thefirst time, describes the isolation of (7E,9E)-ketonitrile 23a as asingle isomer by crystallization in greater than 98% purity withvirtually no contamination from its (7E,9Z)-isomer (23b).

Reduction of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) to(7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19-22. As shown in Scheme 4,(7E,9E)-ketonitrile 23a was reduced to four stereoisomerichydroxynitriles 19-22 with a number of reagents in 92-97% yield and theresults are shown in Table 1. Because (3R,6R)-hydroxynitrile 19 with atrans relationship between the OH at C3 and C6-dienenitrile side chainis the precursor of the naturally occurring (3R,3′R,6′R)-lutein (1), itwas desirable to increase the composition of the trans-hydroxynitriles19 and 20 relative to the cis-hydroxynitriles 21 and 22 in the reductionproducts. The reduction of ketonitrile 23a with NaBH₄ was very sluggishand showed no selectivity with respect to the relative stereochemistryat C3 and C6. This was determined by HPLC analysis of the productsemploying a silica-based nitrile bonded column that allowed theseparation of trans-hydroxynitriles (19+20) from cis-hydroxynitriles(21+22). The reduction products were also monitored by chiral HPLC thatallowed the separation of all four stereoisomers of hydroxynitriles19-22. While the reduction with TIBA was quite efficient even at lowtemperature (−40° C.), the relative composition of trans-hydroxynitriles(19+20) to cis-hydroxynitriles (21+22) could not be dramaticallyaffected. However, the reduction of ketonitrile 23a with a combinationof NaBH₄ and dl-tartaric acid provided trans-hydroxynitriles (19+20) asthe major products (75%) and the cis-hydroxynitriles (21+22) as theminor products (25%). The use of enantiomerically pure d- or l-tartaricacid or their 2,3-dibenzoyl derivatives did not improve thestereoselectivity of this reduction. There are only several reportedexamples of the use of the combination of NaBH₄ and tartaric acid andits derivatives in the reduction of ketones but none of these examplesinvolve the reduction of cyclic α,β-enones (Hirao et al., Agric. Biol.Chem. 1981, 45: 693-697; Adams, Synth. Commun. 1984, 14: 955-959;Yatagai and Ohnuki, J. Chem. Soc. Perkin Trans. 11990, 1826-1828; Cordeset al., Eur. J. Org. Chem. 2005, 24: 5289-5295).

TABLE 1 Reduction of ketonitrile 23a to hydroxynitriles 19-22 withvarious reagents. Temperature (19 + 20):((21 + 22) Reducing agentSolvent (Time, h) (trans:cis)* NaBH₄ EtOH:H₂O 0° C. to R.T. 1:1 1.4:1(24 h) Triisobutylaluminum (TIBA) Toluene −40° C. to R.T. 2:3 (1 h)NaBH₄/dl-Tartaric acid (3/1) EtOH −10 to −15° C. 3:1 (2 h)NaBH₄/d-Tartaric acid (3/1) EtOH −10 to −15° C. 3:1 (2 h)NaBH₄/l-Tartaric acid (3/1) EtOH −10 to −15° C. 3:1 (2 h)NaBH₄/Dibenzoyl-d- EtOH −10 to −15° C. 3:1 tartaric acid (3/1) (2 h)Sodium bis(2-methoxyethoxy)- TBME −5 to 0° C. 1.3:1   aluminum hydride,(1 h) NaAlH₂(OCH₂CH₂OMe)₂ (RED-AL ™) Lithium tri-sec-butylborohydride,TBME −30° C. 1.2:1   LiB[CHMeCH₂CH₃]₃H (0.5 h) (L-SELECTRIDE ™) Sodiumtri-sec-butylborohydride, TBME −30° C. 2.5:1   NaB[CHMeCH₂CH₃]₃H (0.5 h)(N-SELECTRIDE ™) Potassium tri-sec-butylborohydride TBME −30° C. 6:1KB[CHMeCH₂CH₃]₃H (0.5 h) (K-SELECTRIDE ™) Potassiumtrisiamylborohydride, TBME −30 to 0° C. 2.2:1   KB[CHMeCHMe₂]₃H (2 h)(KS-SELECTRIDE ™) BH₃/(R)-2-methyl-CBS- TBME 0° C., 1:6 oxazaborolidine(1.5 h) BH₃/(S)-2-methyl-CBS- TBME 0° C., 1:3 oxazaborolidine (1.5 h)*Indicates the stereochemical relationship between the hydroxyl group atC3 and the dienenitrile side chain at C6.

The reduction of 23a with sodium bis(2-methoxyethoxy)aluminum hydride(RED-AL™) or lithium tri-sec-butylborohydride (L-SELECTRIDE™) producedessentially the same results and did not show a significant preferencefor the formation of trans-hydroxynitriles (19+20). However, when sodiumtri-sec-butylborohydride (N-SELECTRIDE™) or potassiumtri-sec-butylborohydride (K-SELECTRIDE™) were employed as the reducingagents, the relative composition of trans-hydroxynitriles (19+20) tocis-hydroxynitriles (21+22) was 71%:29% and 84%:16%, respectively.

The reduction of 23a with potassium trisiamylborohydride(KS-SELECTRIDE™) did not improve the results obtained with K-SELECTRIDE™and afforded the trans-hydroxynitriles (19+20, 69%) as the majorproducts and cis-hydroxynitriles (21+22, 31%) as the minor products.

Contrary to the results obtained with K-SELECTRIDE™, the reduction ofketonitrile 23a with BH₃/(R)-2-methyl-CBS-oxazaborolidine gavecis-hydroxynitriles (21+22) as the major products (86%) and thetrans-hydroxynitriles (19+20) as the minor products (14%). WhenBH₃/(S)-2-methyl-CBS-oxazaborolidine was used as the reducing agent, thecis-hydroxynitriles (21+22) were still obtained as the major productsbut the stereoselectivity was not as high as that obtained with theR-isomer of CBS-oxazaborolidine.

Therefore, the present invention relates to a stereoselective method forreducing ketonitrile 23a to hydroxynitriles 19-22 in which the ratio oftrans-hydroxynitriles (19+20) to that of cis-hydroxynitriles (21+22) canbe controlled by the use of appropriate reducing agents and can varyfrom 6:1 to 1:6.

Synthesis of optically pure hydroxyaldehydes 15-18 from hydroxynitriles19-22. In one embodiment of the present invention, a mixture of the fourhydroxynitriles 19-22 was reduced with DIBAL-H in dichloromethane to aracemic mixture of hydroxyaldehydes 15-18 in 95% yield. In the followingstep, a mixture of hydroxyaldehydes 15 and 16 was readily separated froma mixture of hydroxyaldehydes 17 and 18 by column chromatography (Scheme5). The direct reduction of ketonitrile 23a to hydroxyaldehydes 15-18could also be accomplished in a one-pot reaction usingK-SELECTRIDE™followed by reduction with DIBAL-H to yield 15+16 (86%) asthe major products and 17+18 (14%) as the minor products.

The racemic mixture of hydroxyaldehydes 15 and 16 were separated byenzyme-mediated acylation with lipase AK (Pseudomonas fluorescens) inrefluxing pentane in the presence of vinyl acetate within 48 h. Whilehydroxyaldehyde 16 was acylated to acetoxyaldehyde 25, hydroxyaldehyde15 remained unreacted. Due to their large difference in their solubilityproperties, 25 and 15 were readily separated by column chromatography.Acetoxyaldehyde 25 was nearly quantitatively hydrolyzed tohydroxyaldehyde 16 with KOH/MeOH at 0° C. to prevent the degradation ofthis sensitive end-group. According to chiral HPLC,(3R,6R)-3-hydroxy-α-ionylideneacetaldehyde (15) and(3S,6S)-3-hydroxy-α-ionylideneacetaldehyde (16) were obtained inenantiomeric excess (ee) of 94% and 93%, respectively.

Employing this overall strategy, the racemic mixture of hydroxyaldehydes17 and 18 were similarly resolved by enzyme-mediated acylation withimmobilized lipase AK (Pseudomonas fluorescens) in refluxing pentane inthe presence of vinyl acetate in 50 h. Hydroxyaldehyde 17 underwentacylation to acetoxyaldehyde 26 while hydroxyaldehyde 18 remainedunreacted (Scheme 5). Separation of 18 and 26 was readily accomplishedby column chromatography. This afforded(3R,6S)-3-hydroxy-α-ionylideneacetaldehyde (18) as a single enantiomerin an ee of 92%. Alkaline hydrolysis of 26 with KOH/MeOH at 0° C.,provided (3S,6R)-3-hydroxy-α-ionylideneacetaldehyde (17) in an ee of91%.

Therefore, all four hydroxyaldehydes 15-18 became accessible in opticalpurities ranging from 91-94%. These hydroxyaldehydes were subsequentlyused in the synthesis of the stereoisomeric luteins 1-4 viaC₂₅-hydroxy-apocarotenals 6-9 as shown in Scheme 6.

Determination of the absolute configuration of C₁₅-hydroxyaldehydes15-18. In an attempt to determine the absolute configuration of the fourC₁₅-hydroxyaldehydes 15-18, a model compound in which thestereochemistry at C3 and C6 is known was needed. Such a model compoundcould be prepared from oxidative cleavage of the polyene chain ofnaturally occurring (3R,3′R,6′R)-lutein in which the stereochemistry inthe ε-end group of this carotenoid at C3′ and C6′ is known to be R. Ithas been well established that the oxidative cleavage (degradation) ofcarotenoids results in the formation of numerous ketones, aldehydes, andacids that are known as apocarotenones, apocarotenals, and apocarotenoicacids, respectively. Our overall strategy for the preparation of a modelcompound by oxidative degradation of (3R,3′R,6′R)-lutein is shown inScheme 7. However, prior to oxidative cleavage of (3R,3′R,6′R)-lutein,the two hydroxyl groups in this carotenoid had to be protected.Therefore, (3R,3′R,6′R)-lutein was first acylated with aceticanhydride/Et₃N/TBME at 50° C. and the resulting (3R,3′R,6′R)-luteindiacetate was then subjected to oxidative degradation with TBHP/bleach.The reaction conditions for this oxidative degradation was similar tothose used in oxidation of α-ionylideneacetonitrile (24a/24b) to3-keto-α-ionylideneacetonitrile (23a/23b) described earlier. The onlyexception was that after the addition of bleach at 0° C., the reactionmixture was allowed to warm up to ambient temperature and stirred for 3h to complete the oxidative cleavage of lutein diacetate (Scheme 7).

After alkaline hydrolysis (KOH/MeOH) followed by column chromatography,HPLC analysis of the partially purified product showed the presence ofnumerous oxidation products of lutein. Among these,(3R,6R)-3-hydroxy-13-apo-ε-caroten-13-one (27) with an ε-end group and(3R)-3-hydroxy-13-apo-β-caroten-13-one (28) with a β-end group were themajor stable products. These were isolated by semipreparative HPLC andfully characterized from their NMR, MS, UV-Vis, and CD spectra.Comparison of the CD and NMR spectra of C₁₈-ketone 27 with those of theindividually purified C₁₅-hydroxyaldehydes 15-18 established theabsolute configuration of these compounds.

Synthesis of Luteins 1-4 via C₂₅-Hydroxy-Apocarotenals 6-9. Thetransformation of hydroxyaldehydes 15-18 to luteins 1-4 is shown inScheme 6. In one embodiment of the present invention, the optically pureC₁₅-hydroxyaldehydes 15-18 were first elongated to their correspondingprotected C₂₅-aldehydes 10-13 by olefination with the protected Wittigsalt 14 in the presence of NaOMe/MeOH at ambient temperature. Aftersolvent evaporation and without isolation of the products, theC₂₅-acetals 10-13 that were obtained as a mixture of all-E and 11Z weredeprotected in dilute aqueous HCl (0.3 N) in acetone to giveC₂₅-aldehydes 6-9 as a mixture of all-E and 11Z in isolated yieldsranging from 75-85%. Under the conditions employed for the deprotectionof acetals 10-13, the hydroxyl group at C3 did not undergo epimerizationand the optical purities of the resulting C₂₅-aldehydes 6-9 were notcompromised. This was confirmed by chiral HPLC of the individuallysynthesized C₂₅-aldehydes. The 11Z-isomers of C₂₅-aldehydes 6-9 could becatalytically isomerized to their corresponding all-E-isomers in thepresence of palladium (II) acetate in refluxing ethyl acetate within 2h. However, in a simplified process, this step was shown to beunnecessary and the isomerization of the 11Z and 11′Z-bonds that areformed by Wittig coupling reactions could be postponed until afterluteins 1-4 were prepared.

As mentioned earlier, the preparation and application of the Wittig salt14 in the total synthesis of carotenoids has been well documented in theliterature but this building block has never been employed for thesynthesis of lutein nor it has been applied to the synthesis of itsprecursors, the C₂₅-acetals 10-13 or C₂₅-hydroxyaldehydes 6-9.

In the final step of the synthesis of luteins, each of theC₂₅-hydroxyaldehydes 6-9 that were prepared as a mixture of all-E and11Z-isomers were allowed to react with the Wittig salt 5 to yield theircorresponding luteins 1-4 as a mixture of all-E and 11Z,11′Z-isomers.Each of the individually prepared E/Z-lutein was then thermallyisomerized to its corresponding all-E isomer in a refluxing solution ofethyl acetate within 4 h. The isolated yields of all-E-luteins 1-4 inthe final step of this synthesis ranged from 65-74%. The Wittig salt 5was prepared according to published procedures (Widmer et al., Helv.Chim. Acta, 1990, 73: 861-867; Soukup et al., Helv. Chim. Acta, 1990,73: 868-873). Similarly, the same strategy described above can also beused to elongate C₂₅-hydroxyaldehydes 6-9 with the S-enantiomer ofWittig salt 5 to synthesize the other four stereoisomers of luteins 1-4;these are: (3S,3′S,6′S)-lutein, (3S,3′R,6′R)-lutein,(3S,3′R,6′S)-lutein, and (3S,3′S,6′R)-lutein.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

Example 1 Synthesis of (rac)-α-Ionylideneacetonitrile (24a/24b) (Route1, Scheme 4)

Methanol (70 mL) was transferred into a 500 mL three-necked flaskequipped with a nitrogen inlet, a thermometer, and an addition funnel.The flask was cooled down in an ice bath under N₂ and sodium (5.47 g,0.238 mol) washed with hexane, was added in small portions bymaintaining the temperature below 10° C. After the sodium was completelydissolved, the solution was stirred at R.T. for 15 minutes and thencooled down to 0° C. A solution of diisopropyl cyanomethylphosphonate(47 g of 95% pure, 44.65 g, 0.218 mol) in TBME (20 mL) was addeddropwise at 0-5° C. in 20 min. The ice bath was removed and the mixturewas allowed to stir at R.T. for 1 h. The reaction mixture was cooleddown in an ice bath and freshly distilled rac-α-ionone (38.10 g, 0.198mol) in TBME (20 mL) was added dropwise in 45 min at 0-5° C. The mixturewas allowed to warm up to room temperature and stirred for 4 h under N₂.The product was quenched with water (100 mL) and the organic layer wasremoved. The aqueous layer was extracted with TBME (2×50 mL) and thecombined organic layer was sequentially washed with brine and water,dried over Na₂SO₄, and evaporated to dryness to give 45.4 g of a paleyellow oil. The crude product was purified by fractional distillation toyield a mixture of 24a and 24b (b.p.=107-110° C. at 10 mm) as acolorless oil (31.6 g, 0.147 mol, 74%) which was shown by ¹H- and¹³C-NMR to consist of an isomeric mixture of 7E,9E:7E,9Z=3:1.

Example 2 Oxidation of (rac)-α-Ionylideneacetonitrile (24a/24b) to(rac)-3-Keto-α-Ionylideneacetonitrile (23a/23b) by Bleach and AqueousTBHP

(rac)-α-Ionylideneacetonitrile (13.15 g, 61.06 mmol) was transferredinto a 500 mL three-necked flask using acetonitrile (30 mL, 23.58 g,0.574 mol). K₂CO₃ (0.844 g, 6.11 mmol) was added and the mixture wascooled down in an ice-salt bath to 0° C. under N₂. A 70% solution ofTBHP in water (52 mL, 46.8 g 70% ≈32.76 g, 0.364 mol) was diluted withacetonitrile (21 mL, 16.51 g, 0.40 mol) and added dropwise to themixture under N₂ at 0° C. in 30 min. Household bleach containing 5.25%NaOCl (260 g, 13.65 g NaOCl, 0.183 mol) was then added over a period of5 h at −5 to 0° C. After the addition was completed, the reactionmixture was stirred at 0° C. for an additional hour. The product wasextracted with hexane (150 mL) and the organic layer was separated. Thewater layer was washed with hexane (2×100 mL) and the combined organiclayer was washed with water (3×150 mL), dried over Na₂SO₄, andevaporated to give 20 g of a yellow oil. The crude product was purifiedby column chromatography (hexane:ethyl acetate, from 98:2 to 92:8) toyield a mixture of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) and(7E,9Z)-3-keto-α-ionylideneacetonitrile (23b) (7.92 g, 34.54 mmol, 57%)as a yellow oil. The product was shown by HPLC (silica-based nitrilebonded column) and ¹H- and ¹³C-NMR to consist of an isomeric mixture of7E,9E:7E,9Z=3:1. Crystallization from ethanol at −20° C. gave 23a as awhite crystal (5.15 g, 22.46 mmol, 37% isolated yield, m.p.=93-95° C.).

Example 3 Palladium(II)-Mediated Oxidation of(rac)-α-Ionylideneacetonitrile (24a/24b) to(rac)-3-Keto-α-Ionylideneacetonitrile (23a/23b) with Anhydrous TBHP

A solution of (rac)-α-Ionylideneacetonitrile (19.60 g, 91.02 mmol) indichloromethane (150 mL) in a 500 mL three-necked flask was cooled downin an ice-salt bath to 0° C. under N₂ and was treated with K₂CO₃ (8.4 g,60.78 mmol) and Pd/C (10 wt. % on C, 7.5 g˜0.75 g Pd, 7.05 mmol). A 5.5M anhydrous solution of TBHP in decane (100 mL, 0.55 mol) was added tothe mixture dropwise while maintaining the temperature at 0° C. Themixture was stirred for 36 h at 0° C. and 50 h at R.T. under N₂. Thesolids were removed by filtration through celite and the filtrate waswashed with water (3×150 mL), brine, and dried over Na₂SO₄. The solventwas removed under reduced pressure to give 24 g of a yellow oil. Thecrude product was purified by column chromatography (hexane:ethylacetate, from 98:2 to 92:8) to yield a mixture of 23a and 23b (11.05 g,48.18 mmol, 53%) as a yellow oil. The product was shown by HPLC and ¹H-and ¹³C-NMR to consist of an isomeric mixture of 7E,9E:7E,9Z=3:1.Crystallization from ethanol at −20° C. gave the (7E,9E)-isomer (23a) asa white crystal (6.00 g, 26.20 mmol, 29% isolated yield).

Example 4 Synthesis of (rac)-α-Ionylideneacetonitrile (24a/24b) byCondensation of α-Ionone with Cyanoacetic Acid (Route 2, Scheme 4)

Freshly distilled rac-α-ionone (32.0 g, 0.166 mol) was transferred intoa 250 mL three necked flask using cyclohexylamine (55 mL, 47.63 g, 480mmol). Cyanoacetic acid (17.85 g, 210 mmol) was added and the mixturewas heated at 80-85° C. under N₂. After 3.5 h, the mixture was allowedto cool down to room temperature and the product was partitioned betweenhexane (150 mL) and water (150 mL). The organic layer was removed andthe aqueous layer was extracted with hexane (50 mL). The combinedorganic layer was washed with water (3×200 mL), dried over Na₂SO₄, andevaporated to dryness to give 33.9 g of a pale yellow oil. The crudeproduct was purified by fractional distillation to yield a mixture of24a and 24b (b.p.=105-110° C. at 10 mm) as a colorless oil (26.66 g,0.124 mol, 75%) that was shown by ¹H- and ¹³C-NMR as well as HPLC toconsist of 24a (92%) and 24b (8%) [ratio of isomeric mixture:7E,9E:7E,9Z=11.5:1].

Example 5 Oxidation of (rac)-α-Ionylideneacetonitrile (24a:24b=11.5:1)to (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) by Bleach and AqueousTBHP

(rac)-α-Ionylideneacetonitrile (26.66 g, 123.8 mmol; 24a:24b=11.5:1) wastransferred into a 1 L three-necked flask using acetonitrile (103 mL,80.96 g, 1.97 mol). K₂CO₃ (1.71 g, 12.37 mmol) was added and the mixturewas cooled down in an ice-salt bath to 0° C. under N₂. A 70% solution ofTBHP in water (124 mL, 111.6 g 70% ≈78.12 g, 867 mmol) was addeddropwise to the mixture under N₂ at 0° C. in 30 min. Household bleachcontaining 5.25% NaOCl (386 g, 20.27 g NaOCl, 272.3 mmol) was then addedover a period of 8 h at −5 to 0° C. After the addition was completed,the reaction mixture was stirred at 0° C. for an additional hour. Theproduct was extracted with hexane (200 mL) and the organic layer wasseparated. The water layer was washed with hexane (2×100 mL) and thecombined organic layer was washed with water (3×200 mL), dried overNa₂SO₄, and evaporated to give 36.7 g of a yellow oil. The crude productwas purified by column chromatography (hexane:ethyl acetate, from 98:2to 92:8) to yield a mixture of (7E,9E)-3-keto-α-ionylideneacetonitrile(23a) and (7E,9Z)-3-keto-α-ionylideneacetonitrile (23b) (15.05 g, 65.63mmol, 53%) as a yellow oil. The product was shown by HPLC (silica-basednitrile bonded column) and ¹H NMR to consist of 23a (92%) and 23b (8%)[ratio of isomeric mixture: 7E,9E:7E,9Z=11.5:1]. Crystallization fromethanol at −20° C. gave 23a as a white crystal (10.5 g, 45.79 mmol, 37%isolated yield, m.p.=93-95° C.).

Example 6 Oxidation of (rac)-α-Ionone to (rac)-3-Keto-α-Ionone by Bleachand Aqueous TBHP (Route 3, Scheme 4)

Freshly distilled (rac)-α-ionone (20.00 g, 104.0 mmol) was transferredinto a 500 mL three-necked flask using EtOAc (103 mL, 92.08 g, 1.05mol). K₂CO₃ (1.44 g, 10.42 mmol) was added and the mixture was cooleddown in an ice-salt bath to 0° C. under N₂. A 70% solution of TBHP inwater (89 mL, 80.1 g 70%≈56.07 g, 0.622 mol) was added dropwise to themixture under N₂ at 0° C. in 30 min. Household bleach containing 5.25%NaOCl (295 g, 15.49 g NaOCl, 0.208 mol) was then added over a period of5 h at −5 to 0° C. After the addition was completed, the reactionmixture was stirred at 0° C. for an additional hour. The organic layerwas removed and the water layer was washed with EtOAc (2×100 mL). Thecombined organic layer was washed with water (2×150 mL), dried overNa₂SO₄, and evaporated to give 26.8 g of a yellow oil. The crude productwas purified by column chromatography (hexane:acetone, from 98:2 to92:8) to yield (rac)-3-keto-α-ionone (13.70 g, 66.41 mmol, 64%).

Example 7 Palladium(II)-Mediated Oxidation of (rac)-α-Ionone to(rac)-3-Keto-α-Ionone with Anhydrous TBHP (Route 3, Scheme 4)

Freshly distilled (rac)-α-ionone (1.00 g, 5.20 mmol) in dichloromethane(10 mL) was cooled down in an ice-salt bath to 0° C. under N₂ and wastreated with K₂CO₃ (0.180 g, 1.30 mmol) and Pd/C (10 wt. % on C, 0.150g˜15 mg Pd, 0.14 mmol). A 5.5 M anhydrous solution of TBHP in decane (5mL, 27.5 mmol) was added to the mixture at 0° C. The mixture was stirredfor 24 h at 0° C. and 12 h at R.T. under N₂. The solids were removed byfiltration through celite and the filtrate was washed with water (3×20mL), brine, and dried over Na₂SO₄. The solvent was removed under reducedpressure to give 1.3 g of a yellow oil. The crude product was purifiedby column chromatography (hexane:ethyl acetone, from 98:2 to 92:8) toyield (rac)-3-keto-α-ionone (0.57 g, 2.76 mmol, 53%).

Example 8 Synthesis of (rac)-3-Keto-α-Ionylideneacetonitrile (23a/23b)from (rac)-3-Keto-α-Ionone

Sodium hydride (0.427 g of 60% suspension in oil ≈0.256 g, 10.67 mmol)was placed in a three-necked flask equipped with a nitrogen inlet and athermometer and washed with hexane (2×10 mL). TBME (30 ml) was added andthe mixture was cooled to 0° C. Diethyl cyanomethylphosphonate (0.964 gof 98% pure, 0.945 g, 5.33 mmol) in 10 mL TBME was added to thesuspension at 5-10° C. under N₂ and the mixture was allowed to stir atR.T. for 1 h. The reaction mixture was cooled down in an ice bath and(rac)-3-keto-ionone (1 g, 4.85 mmol) in 10 mL TBME was added dropwise in30 min at 0-5° C. After stirring for 6 hours at R.T., the reaction wasquenched with water and the organic layer was removed. The aqueous layerwas extracted with TBME (2×20 mL). The combined organic layer was washedwith water, dried over Na₂SO₄, and evaporated to dryness. The crudeproduct (1.1 g) was purified by column chromatography (hexane:acetone,from 98:2 to 95:5) to yield a mixture of 23a and 23b (0.9 g, 3.92 mmol,81%) as a pale yellow oil. The product was shown by HPLC as well as ¹H-and ¹³C-NMR to consist of 23a (75%) and 23b (25%) [ratio of isomericmixture: 7E,9E:7E,9Z=3:1].

Example 9 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) to(7E,9E)-3-Hydroxy-α-Ionylideneacetonitrile (19-22) with NaBH₄

To a solution of (7E,9E)-3-keto-α-ionylideneacetonitrile (2 g, 8.72mmol) in 20 mL ethanol and 15 mL water was added NaBH₄ (0.66 g, 17.45mmol) at 0° C. The mixture was allowed to warm up to room temperature,stirred for 24 h, and the product was partitioned between water (30 mL)and ethyl acetate (50 mL). The organic layer was removed and the aqueouslayer was extracted with 30 mL of ethyl acetate. The combined organiclayer was washed with brine and water, dried over Na₂SO₄, and evaporatedto dryness. The crude product was purified by column chromatography(hexane:acetone=97:3) to afford 3-hydroxy-α-ionylideneacetonitriles19-22 (1.95 g, 8.43 mmol, 97%) as a colorless oil. A mixture of 19+20was separated from 21+22 by semipreparative HPLC and was fullycharacterized by ¹H and ¹³C NMR as well as mass spectrometry andUV-visible spectrophotometry. The isomeric ratio of (19+20):(21+22)=1:1was established by normal phase HPLC (silica-based nitrile bondedcolumn) of the mixture. Hydroxynitriles 19+20 and 21+22 were each shownby chiral HPLC (hexane, 95%; 2-propanol, 5%; CH₃CN, 0.75%) to consist ofan approximately 1:1 mixture of enantiomers.

Example 10 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) to(7E,9E)-3-Hydroxy-α-Ionylideneacetonitrile (19-22) withTriisobutylaluminum (TIBA)

A solution of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) (148 mg,0.65 mmol) in toluene (10 mL) was cooled down to −40° C. under N₂ and asolution of triisobutylaluminum (3 mL of 1M in toluene, 3 mmol) wasadded. The course of the reaction was monitored by HPLC. The mixture wasallowed to warm up to R.T. and stirred for 1 h. The reaction wasquenched by adding a dilute aqueous solution of HCl (0.5 mL, 5% v/v)followed by water (10 mL). The product was diluted with TBME (10 mL) andwashed sequentially with brine and water. The organic layer was driedover Na₂SO₄ and evaporated to dryness. The product (143 mg, 0.62 mmol,95%) was shown by HPLC to consist of two fractions which were separatedby semipreparative HPLC and identified in the order of chromatographicelution (silica-based nitrile bonded column) as(7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (40%) and(7E,9E)-3-hydroxy-α-ionylidene-acetonitriles 21+22 (60%). Theidentification was accomplished by comparison of the ¹H- and ¹³C-NMRspectra as well as HPLC retention times of the hydroxynitriles withthose of authentic samples characterized earlier. Hydroxynitriles 19+20and 21+22 were each shown by chiral HPLC to consist of an approximately1:1 mixture of enantiomers.

Example 11 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) to(7E,9E)-3-Hydroxy-α-Ionylideneacetonitrile (19-22) with SodiumBorohydride/dl-Tartaric acid

A solution of dl-tartaric acid (46 mg, 0.31 mmol) in EtOH (4 mL) wascooled down to 0° C. and was treated with NaBH₄ (12 mg, 0.32 mmol).After the evolution of H₂ subsided, the mixture was stirred at R.T. for1 h and was then cooled down to −15° C. and treated with a solution of(7E,9E)-rac-3-keto-α-ionylideneacetonitrile (23a) (72 mg, 0.31 mmol) inEtOH (3 mL). NaBH₄ (24 mg, 0.63 mmol) in EtOH (3 mL) was added to thesuspension at −15° C. and the course of the reaction was followed byHPLC (silica-based nitrile bonded column). After 2 h, the product waspartitioned between water (10 mL) and ethyl acetate (15 mL). The organiclayer was removed and the aqueous layer was extracted with ethyl acetate(10 mL). The combined organic layer was washed with water (2×10 mL),dried over Na₂SO₄, and evaporated to dryness. The crude product (68.0mg, 0.29 mmol, 94%) was shown by HPLC to consist of two major fractionswhich were separated by semipreparative HPLC and identified in the orderof chromatographic elution as(7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (70%) and(7E,9E)-3-hydroxy-α-ionylidene-acetonitriles 21+22 (30%). The ¹H- and¹³C-NMR as well as HPLC retention times of the hydroxynitriles wereidentical with those of authentic samples of these compoundscharacterized earlier. Hydroxynitriles 19+20 and 21+22 were each shownby chiral HPLC to consist of an approximately 1:1 mixture ofenantiomers.

Example 12 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) to(7E,9E)-3-Hydroxy-α-Ionylideneacetonitrile (19-22) with SodiumBorohydride/2,3-Dibenzoyl-d-Tartaric Acid

A solution of 2,3-dibenzoyl-d-tartaric acid (94 mg, 0.26 mmol) in EtOH(4 mL) was cooled down to 0° C. and was treated with NaBH₄ (10 mg, 0.26mmol). After the evolution of H₂ subsided, the mixture was stirred atR.T. for 1 h and was then cooled down to −15° C. and treated with asolution of (7E,9E)-rac-3-keto-α-ionylideneacetonitrile (23a) (60 mg,0.26 mmol) in EtOH (3 mL). NaBH₄ (20 mg, 0.53 mmol) in EtOH (3 mL) wasadded to the suspension at −15° C. and the course of the reaction wasfollowed by HPLC (silica-based nitrile bonded column). After 2 h, theproduct was partitioned between water (10 mL) and ethyl acetate (15 mL).The organic layer was removed and the aqueous layer was extracted withethyl acetate (10 mL). The combined organic layer was washed with water(2×10 mL), dried over Na₂SO₄, and evaporated to dryness. The crudeproduct (57.8 mg, 0.25 mmol, 96%) was shown by HPLC to consist of amixture of (7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (74%) and(7E,9E)-3-hydroxy-α-ionylidene-acetonitriles 21+22 (26%). The ¹H- and¹³C-NMR spectra as well as HPLC retention times of the hydroxynitrileswere identical with those of authentic samples of these compoundscharacterized earlier. Hydroxynitriles 19+20 and 21+22 were each shownby chiral HPLC to consist of an approximately 1:1 mixture ofenantiomers.

Example 13 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) toHydroxynitrile 19-22 with Sodium Bis(2-Methoxyethoxy)Aluminum Hydride(RED-AL™)

A solution of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) (120 mg,0.524 mmol) in TBME (5 mL) was cooled down to −5° C. under N₂, asolution of Red-Al™ (0.18 mL of 0.65 wt. % in toluene, 0.119 g, 0.59mmol) in TBME (1 mL) was added, and the mixture stirred for 1 h at thistemperature. The reaction was quenched by adding water (10 mL) and theproduct was extracted with TBME (10 mL) and washed sequentially withbrine and water. The organic layer was dried over Na₂SO₄ and evaporatedto dryness. The product (115 mg, 0.497 mmol, 95%) was shown by HPLC(silica-based nitrile bonded column) to consist of(7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (57%) and(7E,9E)-3-hydroxy-α-ionylidene-acetonitriles 21+22 (43%). Theidentification was accomplished by comparison of the HPLC retentiontimes and UV spectra of the hydroxynitriles obtained by a photodiodearray detector with those of authentic samples characterized earlier.Hydroxynitriles 19+20 and 21+22 were each shown by chiral HPLC toconsist of an approximately 1:1 mixture of enantiomers.

Example 14 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) toHydroxynitrile 19-22 with Lithium Tri-Sec-Butylborohydride(L-SELECTRIDE™)

A solution of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) (100 mg,0.436 mmol) in TBME (5 mL) was cooled down to −30° C. under N₂, Asolution of L-SELECTRIDE™(0.52 mL of 1 M in THF, 0.52 mmol) in TBME (1mL) was added by a gas-tight syringe, and the mixture was stirred atthis temperature for 0.5 h. The reaction mixture was treated with 0.5 mLof 3 N NaOH followed by 0.5 mL of 30% H₂O₂ and stirred at R.T. for 30min. The product was extracted with TBME (10 mL) and washed sequentiallywith brine and water, dried over Na₂SO₄, and evaporated to dryness togive a colorless oil. The product (94 mg, 0.406 mmol, 93%) was shown byHPLC (silica-based nitrile bonded column) to consist of(7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (55%) and(7E,9E)-3-hydroxy-α-ionylidene-acetonitriles 21+22 (45%). These wereidentified by comparison of their HPLC retention times and UV spectraobtained by a photodiode array detector with those of authentic samplesof these hydroxynitriles characterized earlier. Hydroxynitriles 19+20and 21+22 were each shown by chiral HPLC to consist of an approximately1:1 mixture of enantiomers.

Example 15 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) toHydroxynitrile 19-22 with Sodium Tri-Sec-Butylborohydride(N-SELECTRIDE™)

A solution of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) (100 mg,0.436 mmol) in TBME (5 mL) was cooled down to −30° C. under N₂, Asolution of N-SELECTRIDE™(0.52 mL of 1 M in THF, 0.52 mmol) in TBME (1mL) was added by a gas-tight syringe, and the mixture was stirred atthis temperature for 0.5 h. The product was worked up as in Example 14to give a mixture of (7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20(71%) and (7E,9E)-3-hydroxy-α-ionylidene-acetonitriles 21+22 (29%) [(94mg, 0.406 mmol, 92%)]. Hydroxynitriles 19+20 and 21+22 were each shownby chiral HPLC to consist of an approximately 1:1 mixture ofenantiomers.

Example 16 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) toHydroxynitrile 19-22 with Potassium Tri-Sec-Butylborohydride(K-SELECTRIDE™)

A solution of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) (3 g, 13.08mmol) in TBME (25 mL) was cooled down to −30° C. under N₂, A solution ofK-SELECTRIDE™(20 mL of 1 M in THF, 20 mmol) in TBME (10 mL) was addeddropwise in 40 min and the mixture was stirred at this temperature for 4h. The reaction mixture was treated with 15 mL of 3 N NaOH followed by15 mL of 30% H₂O₂ and stirred at R.T. for 30 min. The product wasextracted with TBME (10 mL) and washed sequentially with brine andwater, dried over Na₂SO₄, and evaporated to dryness to give a colorlessoil. The product (2.85 g, 12.32 mmol, 94%) was shown by HPLC(silica-based nitrile bonded column) to consist of a mixture of(7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (86%) and(7E,9E)-3-hydroxy-α-ionylidene-acetonitriles 21+22 (14%).Hydroxynitriles 19+20 and 21+22 were each shown by chiral HPLC toconsist of an approximately 1:1 mixture of enantiomers.

Example 17 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) to(7E,9E)-3-Hydroxy-α-Ionylideneacetonitrile (19-22) with(R)-2-Methyl-CBS-oxazaborolidine

To a solution of (R)-2-methyl-CBS-oxazaborolidine (0.3 mL 1M in toluene,0.30 mmol) in TBME (4 mL) was added BH₃.THF (0.3 mL 1M in THF, 0.30mmol) at R.T. under N₂. The mixture was stirred at R.T. for 20 min andwas then cooled down to 0° C. and treated with a solution of(7E,9E)-rac-3-keto-α-ionylideneacetonitrile (23a) (69 mg, 0.30 mmol) inTBME (3 mL). After stirring the reaction mixture for 1.5 h at 0° C.,HPLC (silica-based nitrile bonded column) showed the complete reductionof 23a. The reaction was quenched by slow addition of methanol (1 mL)and the product was diluted with TBME, washed with a saturated solutionof NH₄Cl, followed by 5% NaHCO₃, and then brine. The organic layer waswashed with water (10 mL), dried over Na₂SO₄, and evaporated to dryness.The crude product (66.6 mg, 0.29 mmol, 97%) was shown by HPLC to consistof a mixture of (7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (14%)and (7E,9E)-3-hydroxy-α-ionylideneacetonitriles 21+22 (86%). Thehydroxynitriles 19+20 and 21+22 were each shown by chiral HPLC toconsist of an approximately 1:1 mixture of enantiomers.

Example 18 Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile (23a) to(7E,9E)-3-Hydroxy-α-Ionylideneacetonitrile (19-22) with(S)-2-Methyl-CBS-Oxazaborolidine

To a solution of (R)-2-methyl-CBS-oxazaborolidine (0.3 mL 1M in toluene,0.30 mmol) in TBME (4 mL) was added BH₃.THF (0.3 mL 1M in THF, 0.30mmol) at R.T. under N₂. The mixture was stirred at R.T. for 20 min andwas then cooled down to 0° C. and treated with a solution of(7E,9E)-rac-3-keto-α-ionylideneacetonitrile (23a) (69 mg, 0.30 mmol) inTBME (3 mL). The product was worked up as described above to give acolorless oil (65 mg, 0.28 mmol, 93%) was shown by HPLC to consist of amixture of (7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (25%) and(7E,9E)-3-hydroxy-α-ionylideneacetonitriles 21+22 (75%). Thehydroxynitriles 19+20 and 21+22 were each shown by chiral HPLC toconsist of an approximately 1:1 mixture of enantiomers.

Example 19 Reduction of Hydroxynitriles 19-22 to Hydroxyaldehydes 15-18with DIBAL-H

A solution of hydroxynitriles 19+20 (86%) and 21+22 (14%) [2.31 g, 10mmol] in CH₂Cl₂ (10 mL) was cooled down to −40° C. under N₂ and a 1Msolution of DIBAL-H in CH₂Cl₂ (33 mL, 33 mmol) was added dropwise in onehour. After the addition was completed, the reaction mixture was allowedto stir at −30° C. for 1 h. The mixture was then treated with a veryslow addition of a homogeneous mixture of 26 g of water absorbed onn-silica (0.3 g of water/g of silica) at a rate that the temperatureremained below −10° C. [caution: the addition of silica/water results inrapid elevation of the temperature]. After the addition was completed,the reaction mixture was allowed to stir at 0° C. for 2 h. Na₂SO₄ (3 g)was added and the solids were filtered off and washed with CH₂Cl₂ (20mL). The organic layer was washed with water, dried over Na₂SO₄, andevaporated to dryness to give a pale yellow oil (2.7 g). Columnchromatography (hexane:ethyl acetate, 95:5 to 80:20) of the product gavetwo fractions as 15+16 (1.155 g, 4.93 mmol, 49%) and 17+18 (0.493 g, 2.1mmol, 21%).

Example 20 One-Pot Reduction of (7E,9E)-3-Keto-α-Ionylideneacetonitrile(23a) to Hydroxyaldehydes 15-18 with Potassium Tri-Sec-Butylborohydride(K-SELECTRIDE™) Followed by DIBAL-H

A solution of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) (1.2 g, 5.23mmol) in TBME (10 mL) was cooled down to −30° C. under N₂, A solution ofK-SELECTRIDE™(7.6 mL of 1 M in THF, 7.6 mmol) in TBME (5 mL) was addeddropwise in 30 min and the mixture was stirred at this temperature andthe course of the reaction was monitored by HPLC (silica-based nitrilebonded column). After 2 h, 23a was shown by HPLC to have converted to amixture of (7E,9E)-3-hydroxy-α-ionylideneacetonitriles 19+20 (86%) and(7E,9E)-3-hydroxy-α-ionylideneacetonitriles 21+22 (14%). The reactionmixture was then treated with a 1M solution of DIBAL-H in CH₂Cl₂ (13 mL,13 mmol) dropwise in 30 minutes. After the addition was completed, thereaction mixture was allowed to stir at −20° C. for 3 h. The product wasthen treated with a very slow addition of a homogeneous mixture of 20 gof water absorbed on n-silica (0.5 g of water/g of silica) at a ratethat the temperature remained below −10° C. [caution: the addition ofsilica/water results in rapid elevation of the temperature]. Thereaction mixture was allowed to stir at 0° C. for 2 h. Na₂SO₄ (3 g) wasadded and the solids were filtered off and washed with CH₂Cl₂ (20 mL).The organic layer was washed with water, dried over Na₂SO₄, andevaporated to dryness to give a pale yellow oil (1.9 g). Columnchromatography (hexane:ethyl acetate, 95:5 to 80:20) of the product gavetwo fractions as 15+16 (0.942 g, 4.0 mmol, 77%) and 17+18 (0.077 g, 0.33mmol, 6%).

Example 21 Oxidative Degradation of (3R,3′R,6′R)-Lutein Diacetate to(3R,6R)-3-Hydroxy-13-Apo-α-Caroten-13-One (27) and(3R)-3-Hydroxy-13-Apo-β-Caroten-13-One (28)

Preparation of (3R,3′R,6′R)-Lutein Diacetate. Naturally occurring(3R,3′R,6′R)-lutein was obtained from Kemin Health (Des Moines, Iowa)and converted to (3R,3′R,6′R)-lutein diacetate as follows. A solution of(3R,3′R,6′R)-lutein (3 g, 75% pure ≈2.25 g, 3.96 mmol) in 20 mL of THFwas treated with pyridine (2.5 mL, 2.45 g, 30.97 mmol) and aceticanhydride (2.5 mL, 2.71 g, 26.55 mmol) and the mixture was heated at 45°C. under N₂ overnight. The product was partitioned between water (50 mL)and hexane (50 mL). The organic layer was removed and washedsequentially with 50 mL of aqueous HCl (5%, v/v), 50 mL of saturatedsodium bicarbonate solution, and water (50 mL). The organic layer wasdried over Na₂SO₄ and evaporated to dryness to give a red solid whichwas purified by column chromatography on n-silica (hexane:acetone, from90:10 to 70:30) to give lutein diacetate (2.30 g, 3.52 mmol; 89%).

Oxidative Degradation of (3R,3′R,6′R)-Lutein Diacetate. A solution of(3R,3′R,6′R)-lutein diacetate (1 g, 1.53 mmol) in ethyl acetate (30 mL)was cooled down in an ice-salt bath to 0° C. under N₂ and was treatedwith a 70% solution of TBHP in water (2.70 mL, 2.43 g 70% ≈1.70 g, 18.86mmol). Household bleach containing 5.25% NaOCl (8.84 g, 0.464 g NaOCl,6.23 mmol) was then added over a period of 20 min at 0° C. After theaddition was completed, the reaction mixture was allowed to warm up toR.T. and stirred for 3 h. The organic layer was removed and the waterlayer was washed with EtOAc (2×100 mL). The combined organic layer waswashed with water (2×150 mL), dried over Na₂SO₄, and evaporated todryness. The residue was dissolved in dichloromethane (30 mL) andsaponified with KOH/MeOH (30 mL, 10%, wt/v) at R.T. under N₂. After 2 h,the product was washed with water (3×100 mL), dried over Na₂SO₄, andevaporated to dryness. Purification by column chromatography on n-silica(hexane:acetone, from 95:5 to 70:30) followed by semipreparative HPLC(nitrile bonded column) afforded two major products which were fullycharacterized from their UV-Vis, CD, ¹H- and ¹³C-NMR, and mass spectraas (3R,6R)-3-hydroxy-13-apo-ε-caroten-13-one (27) and(3R)-3-hydroxy-13-apo-α-caroten-13-one (28).

Example 22 Enzyme-Mediated Acylation of(7E,9E)-3-Hydroxy-α-Ionylideneacetaldehydes 15+16 with Lipase AK(pseudomonas fluorescens)

To a solution of (7E,9E)-3-hydroxy-α-ionylideneacetaldehydes 15+16 (2.4g, 10.32 mmol) in 20 mL of pentane was added 1.5 g of lipase AK(pseudomonas fluorescens) and vinyl acetate (2.84 mL, 2.65 g, 30.78mmol). The mixture was refluxed (35-36° C.) under N₂ and the course ofthe enzymatic acylation was monitored by chiral HPLC (2-propanol, 2%;CH₃CN, 98%). After 48 h, the product was filtered through celite and thefiltrate was evaporated to dryness to give a yellow oil (2.7 g). Columnchromatography (hexane:ethyl acetate, 98:2 to 85:15) of the product gavetwo major fractions.

The first fraction was tentatively identified from its ¹H NMR and UVspectrum as (3S,6S)-3-acetoxy-α-ionylideneacetaldehyde (25) [1.22 g,4.41 mmol, 43%]. This fraction was dissolved in CH₂Cl₂ (25 mL) andtreated with KOH/MeOH (2.3 mL, 10% wt/v) for 2 h at 0° C. The productwas washed with water (3×50 mL), dried over Na₂SO₄, and evaporated todryness. The product was fully characterized from its UV, CD, ¹H- and¹³C-NMR, and mass spectra as (3S,6S)-3-hydroxy-α-ionylideneacetaldehyde(16) (1.00 g, 4.27 mmol; 97%). The optical purity of 16 (93% ee) wasestablished by chiral HPLC.

The second fraction was fully characterized from its UV, CD, ¹H- and¹³C-NMR, and mass spectra as (3R,6R)-3-hydroxy-α-ionylideneacetaldehyde(15) (1.03 g, 4.40 mmol, 43%). The optical purity of 15 (94% ee) wasestablished by chiral HPLC.

The absolute configuration of hydroxyaldehydes 15 and 16 was assignedfrom comparison of their ¹H NMR and CD spectra with those of C₁₈-ketone27.

Example 23 Enzyme-Mediated Acylation of(7E,9E)-3-Hydroxy-α-Ionylideneacetaldehydes 17+18 with Lipase AK(pseudomonas fluorescens)

To a solution of (7E,9E)-3-hydroxy-α-ionylideneacetaldehydes 17+18(0.843 g, 3.60 mmol) in 20 mL of pentane was added 0.58 g of lipase AK(pseudomonas fluorescens) and vinyl acetate (1.4 mL, 1.31 g, 15.22mmol). The mixture was refluxed (35-36° C.) under N₂ and the course ofthe enzymatic acylation was monitored by chiral HPLC (2-propanol, 2%;CH₃CN, 98%). After 50 h, the product was filtered through celite and thefiltrate was evaporated to dryness to give a yellow oil (1.0 g). Columnchromatography (hexane:ethyl acetate, 98:2 to 85:15) of the product gavetwo major fractions.

The first fraction was tentatively identified from its ¹H NMR and UVspectrum as (3S,6R)-3-acetoxy-α-ionylideneacetaldehyde (26) [0.319 g,1.15 mmol, 32%]. This fraction was dissolved in CH₂Cl₂ (25 mL) andhydrolyzed with KOH/MeOH (0.8 mL, 10% wt/v) for 2 h at 0° C. The productwas worked up as in Example 21 and was fully characterized from its UV,CD, ¹H- and ¹³C-NMR, and mass spectra as(3S,6R)-3-hydroxy-α-ionylidene-acetaldehyde (17) (0.267 g, 1.14 mmol;99%). The optical purity of 17 (91% ee) was established by chiral HPLC.

The second fraction was fully characterized from its UV, CD, ¹H- and¹³C-NMR, and mass spectra as (3R,6S)-3-hydroxy-α-ionylideneacetaldehyde(18) (0.31 g, 1.32 mmol, 37%). The optical purity of 18 (92% ee) wasestablished by chiral HPLC.

The absolute configuration of hydroxyaldehydes 17 and 18 was assignedfrom comparison of their ¹H NMR and CD spectra with those of C₁₈-ketone27.

Example 24 Preparation of(all-E)-(7-Fomyl-2-Methyl-2,4,6-Octatrienyl)-Triphenyl PhosphoniumChloride Dimethyl Acetal (14)

(all-E)-(7-Fomyl-2-methyl-2,4,6-octatrienyl)triphenyl phosphoniumchloride was prepared according to the method developed by Bernhard etal. (Helv. Chim. Acta 1980, 63: 1473-1490) and was freshly converted toits dimethyl acetal (14) prior to Wittig condensation reactions. Amixture of (all-E)-(7-fomyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium chloride (1.36 g, 3.04 mmol), trimethylorthoformate (0.38 g,3.58 mmol), p-TsOH (6 mg) in methanol (20 mL) was stirred for 3 h at 30°C. The mixture was treated with a few drops of N,N-diisopropylethylamine(DIPEA) and concentrated on a rotary evaporator below 40° C. Theconcentrated solution of the protected Wittig salt 14 (1.42 g, 2.88mmol, 95%) in methanol was directly used in the coupling reactionswithout purification.

Example 25 General Procedure for the Synthesis of C₂₅-hydroxyaldehydes6-9 Synthesis of (3R,6R)-3-Hydroxy-12′-Apo-ε-Caroten-12′-al (6)

A solution of (3R,6R)-3-hydroxy-α-ionylideneacetaldehyde (15) (250.4 mg,1.07 mmol) in MeOH (3 mL) was treated with a solution of the protectedWittig salt 14 (817.7 mg, 1.66 mmol) in methanol (2 mL) at R.T. underN₂. 1 mL of a 0.42 M solution of NaOMe (0.42 mmol) in MeOH (freshlyprepared from Na in MeOH) was added and the mixture was stirred at R.T.for 4 h. The product was partitioned between water (50 mL) and CH₂Cl₂(30 mL), the organic layer was removed, and the water layer wasextracted with CH₂Cl₂ (20 mL). The combined organic layer was washedwith water (2×30 mL), dried over Na₂SO₄, and evaporated to dryness togive a red solid (1.3 g). A small quantity of the solid was purified bysemipreparative HPLC and identified from its UV-visible, ¹H- and¹³C-NMR, and mass spectra as (3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-aldimethyl acetal (10). The red solids were dissolved in acetone (4 mL)and water (1 mL) and stirred with 0.075 mL of 0.3 N HCl for 1 h at R.T.under N₂. The product was extracted with CH₂Cl₂, and sequentially washedwith saturated solution of NaHCO₃ and water, dried over Na₂SO₄, andevaporated to dryness to give a red oil. Column chromatography(hexane:ethyl acetate, 95:5 to 80:20) gave a red solid that wasidentified from its UV-visible, CD, ¹H- and ¹³C-NMR, and mass spectra as(3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (6) (334 mg, 0.91 mmol; 85%).

Following the above procedure,(3S,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (7),(3S,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (8), and(3R,6S)-3-hydroxy-12′-apo-α-caroten-12′-al (9) were prepared in yieldsranging from 75-85%.

Example 26 General Procedure for the Synthesis of Luteins 14 Synthesisof (3R,3′R,6′R)-Lutein (1)

A solution of (3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (6) (257 mg,0.70 mmol) and (3R)-3-hydroxy-(α-ionylideneethyl)triphenylphosphoniumchloride (Wittig salt 5) (410 mg, 0.79 mmol) in CH₂Cl₂ (5 mL) was cooleddown to −5° C. under N₂. A solution of KOH (130 mg, 2.32 mmol) in H₂O(0.5 mL) was added and the mixture was stirred for 0.5 h at −5° C. and 3h at R.T. Dichloromethane (20 mL) was added, and the product was washedwith water (3×10 mL). The organic layer was removed, dried over Na₂SO₄,and evaporated to dryness to give 1 g of a red oil. The crude productwas then refluxed in ethyl acetate for 4 h under N₂ to affect the cis(z) to trans (E) thermal isomerization of lutein. After solventevaporation, the product was purified by column chromatography(hexane:ethylacetate, from 90:10 to 50:50) to give a red solid that wascrystallized from hexane:acetone=4:1 and identified from its UV-visible,CD, ¹H- and ¹³C-NMR, and mass spectra as (3R,3′R,6′R)-Lutein (1) (0.294g, 0.517 mmol; 74%).

Following the above procedure, (3R,3′S,6′S)-lutein (2),(3R,3′S,6′R)-lutein or 3′-epilutein (3), and (3R,3′R,6′S)-lutein (4)were prepared in yields ranging from 65-74%.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1. A method for synthesis of a compound having the Formula (I):

comprising reacting a compound having the Formula (II):

with a compound having the Formula (III):

via Wittig coupling, wherein A⊖ is an anionic counterion.
 2. The methodof claim 1, wherein said compound of Formula (I) is (3R,3′R,6′R)-lutein,(3R,3′S,6′S)-lutein, (3R,3′S,6′R)-lutein, (3R,3′R,6′S)-lutein,(3S,3′S,6′S)-lutein, (3S,3′R,6′R)-lutein, (3S,3′R,6′S)-lutein or(3S,3′S,6′R)-lutein or a combination thereof.
 3. The method of claim 1,wherein said compound of Formula (III) is(3R)-3-hydroxy-(β-ionylideneethyl)triphenylphosphonium salt or(3S)-3-hydroxy-(β-ionylideneethyl)triphenylphosphonium salt.
 4. Themethod of claim 3, wherein said salt is the chloride, bromide or iodidesalt.
 5. The method of claim 1, wherein the compound of Formula II isprepared by a process comprising deprotecting a compound having theFormula (IV):

to obtain said compound having the Formula II, wherein R¹ and R² areindependently a branched C₁-C₇ alkyl, a straight chain C₁-C₇ alkyl, ortaken together form a 5-7 membered ring.
 6. The method of claim 5,wherein said compound having the Formula (IV) is deprotected under mildacidic conditions without loss of optical purity.
 7. The method of claim5, wherein R¹ and R² are independently C₁-C₇ alkyl.
 8. The method ofclaim 7, wherein R¹ and R² are methyl.
 9. The method of claim 5, whereinsaid compound having the Formula (II) is(3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (6),(3S,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (7),(3S,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (8) or(3R,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (9), or a combinationthereof.
 10. The method of claim 9, wherein (i)(3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al dimethylacetal (10) isdeprotected to form (3R,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (6), (ii)(3S,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al dimethylacetal (11) isdeprotected to form 3S,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (7); (iii)(3S,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al dimethyl acetal (12) isdeprotected to form (3S,6R)-3-hydroxy-12′-apo-ε-caroten-12′-al (8) or(iv) 3R,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al dimethylacetal (13) isdeprotected to form (3R,6S)-3-hydroxy-12′-apo-ε-caroten-12′-al (9). 11.The method of claim 5, wherein the compound of Formula IV is prepared bya process comprising elongating a compound having the Formula V:

with a compound having the Formula VI:

via Wittig coupling to obtain said compound having the Formula IV, whereX⊖ is an anionic counterion, wherein R³ and R⁴ are independently abranched C₁-C₇ alkyl, a straight chain C₁-C₇ alkyl, or taken togetherform a 5-7 membered ring.
 12. The method of claim 11, wherein R¹ and R²are independently C₁-C₇ alkyl.
 13. The method of claim 12, wherein R¹and R² are methyl.
 14. The method of claim 11, wherein said compoundhaving the Formula (IV) is protected C₂₅-hydroxyaldehyde 10, 11, 12 or13, or a combination thereof.
 15. The method of claim 11, wherein saidcompound having the Formula (VI) is(all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium salt.16. The method of claim 15, wherein said salt is the chloride, bromideor iodide salt.
 17. The method of claim 14, wherein (i)(all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium saltis reacted with (7E,9E,3R,6R)-3-hydroxy-α-ionylideneacetaldehyde (15) toobtain protected C₂₅-hydroxyaldehyde 10; (ii)(all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium saltis reacted with (7E,9E,3S,6S)-3-hydroxy-α-ionylideneacetaldehyde (16) toobtain protected C₂₅-hydroxyaldehyde 11; (iii)(all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium saltis reacted with (7E,9E,3S,6R)-3-hydroxy-α-ionylideneacetaldehyde (17) toobtain protected C₂₅-hydroxyaldehyde 12 or (iv)(all-E)-(7-formyl-2-methyl-2,4,6-octatrienyl)triphenylphosphonium saltis reacted with (7E,9E,3R,6S)-3-hydroxy-α-ionylideneacetaldehyde (18) toobtain protected C₂₅-hydroxyaldehyde
 13. 18. The method of claim 11,wherein the compound of Formula V is prepared by a process comprisingreacting the cyano group of a compound having the Formula VII:

with a reducing agent to obtain said compound having the Formula V:


19. The method of claim 18, wherein said compound having the Formula (V)is C₁₅-hydroxyaldehyde 15, 16, 17 and 18, or a combination thereof. 20.The method of claim 18, wherein said compound having the Formula (VII)is (3R,6R)-3-hydroxy-α-ionylideneacetonitrile (19),(3S,6S)-3-hydroxy-α-ionylideneacetonitrile (20),(3S,6R)-3-hydroxy-α-ionylideneacetonitrile (21) or(3R,6S)-3-hydroxy-α-ionylideneacetonitrile (22), or a combinationthereof.
 21. The method of claim 20, wherein a mixture ofC₁₅-hydroxynitriles (3R,6R)-3-hydroxy-α-ionylideneacetonitrile (19),(3S,6S)-3-hydroxy-α-ionylideneacetonitrile (20),(3S,6R)-3-hydroxy-α-ionylideneacetonitrile (21) and(3R,6S)-3-hydroxy-α-ionylideneacetonitrile (22) is reduced withdiisobutylaluminum hydride (DIBAL-H), to obtain a mixture ofC₁₅-hydroxyaldehydes 15, 16, 17 and
 18. 22. The method of claim 21,further comprising separating a mixture of C₁₅-hydroxyaldehyde 15, 16,17 or 18, or a combination thereof using a combination of columnchromatography and enzyme-mediated acylation.
 23. The method of claim22, wherein a mixture of C₁₅-hydroxyaldehydes 15 and 16 is separatedfrom said mixture of C₁₅-hydroxyaldehyde 15, 16, 17 and 18 by silica gelchromatography using a combination of a hydrocarbon solvent selectedfrom the group consisting of pentane, hexane, heptane and cyclohexane,and ethyl acetate or acetone, to obtain a mixture ofC₁₅-hydroxyaldehydes 15 and
 16. 24. The method of claim 22, wherein amixture of C₁₅-hydroxyaldehydes 17 and 18 is separated from said mixtureof C₁₅-hydroxyaldehyde 15, 16, 17 and 18 by silica gel chromatographyusing a combination of a hydrocarbon solvent selected from the groupconsisting of pentane, hexane, heptane and cyclohexane, and ethylacetate or acetone, to obtain a mixture of C₁₅-hydroxyaldehydes 17 and18.
 25. The method of claim 23, further comprising acylating saidmixture of C₁₅-hydroxyaldehydes 15 and 16 with lipase AK (Pseudomonasfluorescens) or lipase PS (Pseudomonas cepacia) in the presence of anacyl donor, wherein C₁₅-hydroxyaldehyde 16 is converted to(3S,6S)-3-acetoxy-α-ionylideneacetaldehyde (25) whileC₁₅-hydroxyaldehyde 15 remains unesterified.
 26. The method of claim 25,wherein said acyl donor is vinyl acetate.
 27. The method of claim 25,further comprising saponifying C₁₅-acetoxyaldehyde 25 with alcoholicpotassium hydroxide (KOH) or sodium hydroxide (NaOH) to obtainC₁₅-hydroxyaldehyde
 16. 28. The method of claim 24, further comprisingacylating said mixture of C₁₅-hydroxyaldehydes 17 and 18 with lipase AK(Pseudomonas fluorescens) or lipase PS (Pseudomonas cepacia) in thepresence of an acyl donor, wherein C₁₅-hydroxyaldehyde 17 is convertedto (3S,6R)-3-acetoxy-α-ionylideneacetaldehyde (26) whileC₁₅-hydroxyaldehyde 18 remains unesterified.
 29. The method of claim 28,wherein said acyl donor is vinyl acetate.
 30. The method of claim 28,further comprising saponifying C₁₅-acetoxyaldehyde 26 with alcoholicpotassium hydroxide (KOH) or sodium hydroxide (NaOH) to obtainC₁₅-hydroxyaldehyde
 17. 31. The method of claim 18, further comprisingreducing the ketone group of a compound having the Formula (VIII):

with a reducing agent, to obtain said compound having the Formula (VII):


32. The method of claim 31, wherein said compound having the Formula(VIII) is (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) or(7E,9z)-3-keto-α-ionylideneacetonitrile (23b), or a combination thereof.33. The method of claim 31, wherein said compound having the Formula(VII) is (3R,6R)-3-hydroxy-α-ionylideneacetonitrile (19),(3S,6S)-3-hydroxy-α-ionylideneacetonitrile (20),(3S,6R)-3-hydroxy-α-ionylideneacetonitrile (21) or(3R,6S)-3-hydroxy-α-ionylideneacetonitrile (22), or a combinationthereof.
 34. The method of claim 31, wherein said reducing agent isstereoselective.
 35. The method of claim 32, wherein(7E,9E)-3-keto-α-ionylideneacetonitrile (23a) is stereoselectivelyreduced with a reducing agent to obtain (3,6-trans)-C₁₅-hydroxynitriles19+20 and (3,6-cis)-C₁₅-hydroxynitriles 21+22 in a ratio ranging from6:1 to 1:6.
 36. The method of claim 35, wherein said reducing agent isNaBH₄, NaBH₄/dl-tartaric acid, NaBH₄/d-tartaric acid, NaBH₄/l-tartaricacid, NaBH₄/dibenzoyl-d-tartaric acid, NaAlH₂(OCH₂CH₂OMe)₂ (RED-AL™),LiB[CHMeCH₂CH₃]₃H (L-SELECTRIDE™), NaB[CHMeCH₂CH₃]₃H(N-SELECTRIDE™),KB[CHMeCH₂CH₃]₃H (K-SELECTRIDE™), KB[CHMeCHMe₂]₃H (KS-SELECTRIDE™),BH₃/(R)-2-methyl-CBS-oxazaborolidine orBH₃/(S)-2-methyl-CBS-oxazaborolidine.
 37. The method of claim 36,wherein ketonitrile 23a is selectively reduced with KB[CHMeCH₂CH₃]₃H(K-SELECTRIDE™) to obtain (3,6-trans)-C₁₅-hydroxynitriles 19+20 as themajor products and (3,6-cis)-C₁₅-hydroxynitriles 21+22 as the minorproducts.
 38. The method of claim 36, wherein ketonitrile 23a isselectively reduced with BH₃/(R)-2-methyl-CBS-oxazaborolidine to obtain(3,6-cis)-C₁₅-hydroxynitriles 21+22 as the major products and(3,6-trans)-C₁₅-hydroxynitriles 19+20 as the minor products.
 39. Themethod of claim 32, further comprising (i) reducing(7E,9E)-3-keto-α-ionylideneacetonitrile (23a) with a metal hydridereagent to form a mixture of C₁₅-hydroxynitriles 19, 20, 21 and 22 and(ii) reducing said mixture of C₁₅-hydroxynitriles 19, 20, 21 and 22 withDIBAL-H to obtain a mixture of C₁₅-hydroxyaldehydes 15, 16, 17 and 18,in a one-pot reaction.
 40. The method of claim 39, wherein said metalhydride reagent is NaAlH₂(OCH₂CH₂OMe)₂ (RED-AL™), LiB[CHMeCH₂CH₃]₃H(L-SELECTRIDE™), NaB[CHMeCH₂CH₃]₃H(N-SELECTRIDE™), KB[CHMeCH₂CH₃]₃H(K-SELECTRIDE™) or KB[CHMeCHMe₂]₃H (KS-SELECTRIDE™).
 41. The method ofclaim 31, wherein the compound of Formula VIII is prepared by a processcomprising reacting a compound having the Formula (IX):

with an oxidizing agent via allylic oxidation, to obtain said compoundhaving the Formula (VIII):


42. The method of claim 41, wherein said compound having the Formula(IX) is (7E,9E)-α-ionylideneacetonitrile (24a) or(7E,9Z)-α-ionylideneacetonitrile (24b), or a combination thereof. 43.The method of claim 41, wherein said compound having the Formula (VIII)is (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) or(7E,9z)-3-keto-α-ionylideneacetonitrile (23b), or a combination thereof.44. The method of claim 42, wherein a mixture of(7E,9E)-α-ionylideneacetonitrile (24a) and(7E,9Z)-α-ionylideneacetonitrile (24b) in an isomeric ratio ranging from3:1 to 12:1 is reacted with an oxidizing reagent, to obtain a mixture of(7E,9E)-3-keto-α-ionylideneacetonitrile (23a) and(7E,9Z)-3-keto-α-ionylideneacetonitrile (23b) in an isomeric ratioranging from 3:1 to 12:1.
 45. The method of claim 44, further comprisingseparating said mixture of ketonitrile 23a and ketonitrile 23b viacrystallization with an alcohol, at a temperature ranging from −15 to 0°C.
 46. The method of claim 45, wherein said alcohol is ethanol.
 47. Themethod of claim 44, wherein said compound of Formula IX is a mixture of(7E,9E)-α-ionylideneacetonitrile (24a) and(7E,9Z)-α-ionylideneacetonitrile (24b) in an isomeric ratio ranging from3:1 to 12:1 and is oxidized with a combination of tert-BuOOH (TBHP) andbleach (5.25% NaOCl), at a temperature ranging from −5 to 0° C., in asolvent selected from the group consisting of acetonitrile (CH₃CN),methylene chloride (CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran(THF), tert-butyl methyl ether (TBME), dimethylformamide (DMF),dimethylsulfoxide (DMSO), ethylene glycol, a straight chain C₁-C₅alcohol and a branched C₁-C₅ alcohol, to obtain the compound of FormulaVIII as a mixture of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) and(7E,9z)-3-keto-α-ionylideneacetonitrile (23b).
 48. The method of claim44, wherein said compound of Formula IX is a mixture of(7E,9E)-α-ionylideneacetonitrile (24a) and(7E,9Z)-α-ionylideneacetonitrile (24b) in an isomeric ratio ranging from3:1 to 12:1 and is oxidized with a combination of tert-BuOOH (TBHP) andPd/C at a temperature ranging from 0° C. to room temperature (R.T.), ina solvent selected from the group consisting of acetonitrile (CH₃CN),methylene chloride (CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran(THF), tert-butyl methyl ether (TBME), dimethylformamide (DMF),dimethylsulfoxide (DMSO), ethylene glycol, a straight chain C₁-C₅alcohol and a branched C₁-C₅ alcohol, to obtain the compound of FormulaVIII as a mixture of (7E,9E)-3-keto-α-ionylideneacetonitrile (23a) and(7E,9Z)-3-keto-α-ionylideneacetonitrile (23b).
 49. The method of claim41, wherein the compound of Formula IX is prepared by a processcomprising condensing a compound having the Formula (X):

with cyanoacetic acid to obtain said compound having the Formula (IX):


50. The method of claim 49, wherein said compound having the Formula(IX) is (7E,9E)-α-ionylideneacetonitrile (24a) or(7E,9Z)-α-ionylideneacetonitrile (24b), or a mixture thereof.
 51. Themethod of claim 49, wherein said compound having the Formula (X) is(rac)-α-ionone.
 52. The method of claim 51, (rac)-α-ionone is condensedwith cyanoacetic acid in the presence of an amine, at a temperatureranging from 80° C. to 100° C., to obtain(7E,9E)-α-ionylidene-acetonitrile (24a) and(7E,9Z)-α-ionylideneacetonitrile (24b) in a ratio of 12:1 or greater.53. The method of claim 52, wherein said amine is cyclohexylamine. 54.The method of claim 52, further comprising purifying said mixture ofnitrites 24a and 24b in an isomeric ratio of 12:1 or greater by vacuumdistillation, wherein said isomeric ratio of 24a and 24b is unaltered.55. A method comprising reacting (rac)-α-ionone with an oxidizing agentto obtain (rac)-3-keto-α-ionone.
 56. The method of claim 55, wherein(rac)-α-ionone is reacted with a combination of tert-BuOOH (TBHP) andbleach, at a temperature ranging from −5 to 0° C., in a solvent selectedfrom the group consisting of acetonitrile (CH₃CN), methylene chloride(CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran (THF), tert-butylmethyl ether (TBME), dimethylformamide (DMF), dimethylsulfoxide (DMSO),ethylene glycol, a straight chain C₁-C₅ alcohol and a branched C₁-C₅alcohol, to obtain to (rac)-3-keto-α-ionone.
 57. The method of claim 55,wherein (rac)-α-ionone is reacted with a combination of tert-BuOOH(TBHP) and Pd/C, at a temperature ranging from 0° C. to room temperature(R.T.), in a solvent selected from the group consisting of acetonitrile(CH₃CN), methylene chloride (CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran (THF), tert-butyl methyl ether (TBME), dimethylformamide (DMF),dimethylsulfoxide (DMSO), ethylene glycol, a straight chain C₁-C₅alcohol and a branched C₁-C₅ alcohol, to obtain to(rac)-3-keto-α-ionone.
 58. The method of claim 55, further comprisingcondensing said (rac)-3-keto-α-ionone with (EtO)₂P(O)CH₂CN or(iso-PrO)₂P(O)CH₂CN in the presence of a base to obtain ketonitriles 23aand 23b.
 59. A method of preparing(3R,6R)-3-hydroxy-13-apo-ε-caroten-13-one (27) and(3R)-3-hydroxy-13-apo-β-caroten-13-one (28) comprising oxidativelydegrading (3R,3′R,6′R)-lutein diacetate with tert-BuOOH (TBHP) andbleach, at a temperature ranging from −5° C. to room temperature (R.T.),in a solvent selected from the group consisting of acetonitrile (CH₃CN),methylene chloride (CH₂Cl₂), ethyl acetate, hexane, tetrahydrofuran(THF), tert-butyl methyl ether (TBME), dimethylformamide (DMF),dimethylsulfoxide (DMSO), a straight chain C₁-C₅ alcohol and a branchedC₁-C₅ alcohol to obtain (3R,6R)-3-hydroxy-13-apo-ε-caroten-13-one (27)and (3R)-3-hydroxy-13-apo-β-caroten-13-one (28).
 60. A method ofpreparing a compound of the Formula XII and a compound of the FormulaXIII comprising oxidatively degrading a compound having the Formula XI:

with an oxidizing agent, to obtain a compound of the Formula XII:

and a compound of the Formula XIII:


61. The method of claim 60, wherein said compound having the Formula(XI) is (3R,3′R,6′R)-lutein diacetate, (3R,3′S,6′S)-lutein diacetate,(3R,3′S,6′R)-lutein diacetate, (3R,3′R,6′S)-lutein diacetate,(3S,3′S,6′S)-lutein diacetate, (3S,3′R,6R)-lutein diacetate,(3S,3′R,6′S)-lutein diacetate or (3S,3′S,6′R)-lutein diacetate or acombination thereof.
 62. The method of claim 58, wherein(3R,3′R,6′R)-lutein diacetate is oxidatively degraded with tert-BuOOH(TBHP) and bleach, at a temperature ranging from −5° C. to roomtemperature (R.T.), in a solvent selected from the group consisting ofacetonitrile (CH₃CN), methylene chloride (CH₂Cl₂), ethyl acetate,hexane, tetrahydrofuran (THF), tert-butyl methyl ether (TBME),dimethylformamide (DMF), dimethylsulfoxide (DMSO), a straight chainC₁-C₅ alcohol and a branched C₁-C₅ alcohol to obtain(3R,6R)-3-hydroxy-13-apo-ε-caroten-13-one (27) and(3R)-3-hydroxy-13-apo-β-caroten-13-one (28).